Elicitor peptides having disrupted hypersensitive response box and use thereof

ABSTRACT

Disclosed are peptides that induce an active plant response, but not a hypersensitive response, when applied to plant tissue. These peptides also preferably exhibit improved solubility, stability, resistance to chemical degradation, or a combination of these properties. Use of these peptides or fusion polypeptides, or DNA constructs encoding the same, for modulating plant biochemical signaling, imparting disease resistance to plants, enhancing plant growth, imparting tolerance to biotic stress, imparting tolerance and resistance to abiotic stress, imparting desiccation resistance to cuttings removed from ornamental plants, imparting post-harvest disease or post-harvest desiccation resistance to a fruit or vegetable, or enhancing the longevity of fruit or vegetable ripeness are also disclosed.

This application is a continuation of U.S. patent application Ser. No.14/872,347, filed Oct. 1, 2015, which claims the priority benefit ofU.S. Provisional Patent Application Ser. No. 62/058,535, filed Oct. 1,2014, and U.S. Provisional Patent Application Ser. No. 62/186,527, filedJun. 30, 2015, each of which is hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to novel elicitor peptides havingdisrupted hypersensitive response boxes and their use for inducingactive plant responses including, among others, growth enhancement,disease resistance, pest or insect resistance, and stress resistance.

BACKGROUND OF THE INVENTION

The identification and isolation of harpin proteins came from basicresearch at Cornell University attempting to understand how plantpathogenic bacteria interact with plants. A first line of defense is thehypersensitive response (HR), a localized plant cell death at the siteof infection. Cell death creates a physical barrier to movement of thepathogen and in some plants dead cells can release compounds toxic tothe invading pathogen. Research had indicated that pathogenic bacteriawere likely to have a single factor that was responsible for triggeringthe HR. A basic aim of the Cornell research was to identify a specificbacterial protein responsible for eliciting the HR. The target proteinwas known to be encoded by one of a group of bacteria genes called theHypersensitive Response and Pathogenicity (hrp) gene cluster. The hrpcluster in the bacterium Erwinia amylovora (Ea), which causes fireblight in pear and apple, was dissected and a single protein wasidentified that elicited HR in certain plants. This protein was giventhe name harpin (and, later, harpin_(Ea)) and the corresponding genedesignated hrpN. This was the first example of such a protein and geneidentified from any bacterial species.

A number of different harpin proteins have since been identified fromErwinia, Pseudomonas, Ralstonia, Xanthomonas, and Pantoea species, amongothers. Harpin proteins, while diverse at the primary amino acidsequence level, share common biochemical and biophysical characteristicsas well as biological functions. Based on their unique properties, theharpin proteins are regarded in the literature as belonging to a singleclass of proteins.

Subsequent to their identification and isolation, it was thereafterdiscovered that harpins could elicit disease resistance in plants andincrease plant growth. An important early finding was that applicationof purified harpin protein made a plant resistant to a subsequentpathogen attack, and in locations on the plant well away from theinjection site. This meant that harpin proteins can trigger a SystemicAcquired Resistance (SAR), a plant defense mechanism that providesresistance to a variety of viral, bacterial, and fungal pathogens.

In crop protection, there is a continuous need for compositions thatimprove the health of plants. Healthier plants are desirable since theyresult in better yields and/or a better quality of the plants or crops.Healthier plants also better resist biotic and abiotic stress. A highresistance against biotic stresses in turn allows the growers to reducethe quantity of pesticides applied and consequently to slow down thedevelopment of resistances against the respective pesticides.

Harpin_(αβ) is a fusion protein that is derived from several differentharpins. Harpin_(αβ) has been shown to suppress nematode egg production,enhance the growth, quality and yield of a plant, and increase a plant'svigor. Its amino acid and nucleotide sequences are described in detailin U.S. Application Publ. No. 2010/0043095.

To date, harpin and harpin_(αβ) production and their use in agriculturaland horticultural applications have been as a powdered solid coated onstarch. This limits the use and versatility of the harpin proteins,because liquid suspensions of the powdered harpin proteins in water havean effective useful life of only 48-72 hours before significantdegradation and loss of activity occurs. Another problem with harpinsolutions is protein solubility and stability.

It would be desirable to identify synthetic and derivative harpinpeptides that are readily soluble in aqueous solution, stable, resistantto chemical degradation, and effective in inducing active plantresponses that include, among others, enhanced plant growth andproduction, as well as resistance to abiotic and biotic stressors.

The present invention is directed to overcoming these and otherlimitations in the art.

SUMMARY OF THE INVENTION

A first aspect of the invention relates to an isolated peptidecomprising the amino acid sequence of:

J-X-X-X-J-J-X-X-X-J-J-X-X-X-J-J (SEQ ID NO: 1) wherein

the peptide is free of cysteine and methionine;

each X at positions 2, 3, 7, 8, 12, and 13 is optional and, whenpresent, is any amino acid;

each X at positions 4, 9, and 14 is any amino acid;

one to three of the J residues at positions 1, 5, 6, 10, 11, 15, and 16is a non-hydrophobic amino acid or A, and all other of the J residuesare L, I, V, or F, and the peptide induces an active plant response, butdoes not induce a hypersensitive response, when applied to plant tissue.

A second aspect of the invention relates to an isolated peptide havingthe amino acid sequence of:

XXGISEKXXXXXXXXXXXXXXXX (SEQ ID NO: 2, modified P1/P4 consensus),wherein

-   -   X at position 1 is optional and can be S, N, D, isoD, G, A, or        S;    -   X at position 2 is optional and can be Q, E, g-glutamate, G, A,        or S;    -   X at position 8 is Q, E, g-glutamate, G, A, or S;    -   X at position 9 is M, L, I, F, or V, or a non-hydrophobic amino        acid;    -   X at position 10 is optional and can be D or isoD;    -   X at position 11 is Q, E, g-glutamate, G, A, or S;    -   X at position 12 is M, L, I, or F, or a non-hydrophobic amino        acid;    -   X at position 13 is M, L, or I, or a non-hydrophobic amino acid;    -   X at position 14 is optional and can be any hydrophilic amino        acid, preferably S, T, D, isoD, K, or Q, and optionally A or C;    -   X at position 15 is Q, E, g-glutamate, G, A, S, K, or I;    -   X at position 16 is M, L, I, V, or F, or a non-hydrophobic amino        acid;    -   X at position 17 is M, L, I, A, or V, or a non-hydrophobic amino        acid;    -   X at position 18 is Q, E, g-glutamate, G, A, S, M, T, or K;    -   X at position 19 is A, D, isoD, S, V, T, K, R, E, H, or G;    -   X at position 20 is M, L, or I;    -   X at position 21 is M, L, I, V, S, or F, or a non-hydrophobic        amino acid other than serine;    -   X at position 22 is Q, E, g-glutamate, G, A, S;    -   X at position 23 is P, Q, E, g-glutamate, G, A, or S; and        wherein at least one of the residues at positions 9, 12, 13, 16,        17, and 20 is a non-hydrophobic amino acid, or the residue at        position 21 is a non-hydrophobic amino acid other than serine;        and wherein the peptide induces an active plant response, but        does not induce a hypersensitive response, when applied to plant        tissue.

A third aspect of the invention relates to an isolated peptide havingthe amino acid sequence of:

SXGISEKXXDXXXXXXXXAXXXP (SEQ ID NO:3, modified P4 consensus), wherein

-   -   X at position 2 is Q, E, g-glutamate, G, A, or S;    -   X at position 8 is Q, E, g-glutamate, G, A, or S;    -   X at position 9 is M, S, L, A, I, V, or F, or a non-hydrophobic        amino acid other than serine;    -   X at position 11 is Q, E, g-glutamate, G, A, or S;    -   X at position 12 is L, I, or F, or a non-hydrophobic amino acid;    -   X at position 13 is L, A, I, V, or F, or a non-hydrophobic amino        acid;    -   X at position 14 is any hydrophilic amino acid;    -   X at position 15 is Q, E, g-glutamate, G, A, S, K, or I;    -   X at position 16 is L, A, I, V, M, or F, or a non-hydrophobic        amino acid;    -   X at position 17 is M, I, S, or F, or a non-hydrophobic amino        acid other than serine;    -   X at position 18 is Q, E, g-glutamate, G, A, or S;    -   X at position 20 is M, L, I, V, or F, or a non-hydrophobic amino        acid;    -   X at position 21 is M, L or F, or a non-hydrophobic amino acid;        and    -   X at position 22 is Q, E, g-glutamate, G, A, or S,        wherein one of the residues at positions 9, 12, 13, 16, 20, and        21 is a non-hydrophobic amino acid or A, or the residue at        position 17 is a non-hydrophobic amino acid other than serine;        and wherein the peptide induces an active plant response, but        does not induce a hypersensitive response, when applied to plant        tissue.

A fourth aspect of the invention relates to an isolated peptide havingthe amino acid sequence of:

XXGISEKXJDXJJTXJJXAJJXX (SEQ ID NO: 4, modified P1 consensus), wherein

-   -   X at position 1 is N, D, isoD, G, A, or S;    -   X at position 2 is Q, E, g-glutamate, G, A, or S;    -   X at position 8 is Q, E, g-glutamate, G, A, or S;    -   X at position 11 is Q, E, g-glutamate, G, A, or S;    -   X at position 15 is Q, E, g-glutamate, G, A, or S;    -   X at position 18 is M, T, K, E, g-glutamate, G, A, or S;    -   X at position 22 is Q, E, g-glutamate, G, A, or S;    -   X at position 23 is Q, E, g-glutamate, G, A, or S;        J at positions 9, 12, 13, 16, 17, 20, and 21 are hydrophobic        amino acids selected from L, V, I, and F, except that one of the        amino acids at these positions is a non-hydrophobic amino acid        or A;        wherein the peptide induces an active plant response, but does        not induce a hypersensitive response, when applied to plant        tissue.

A fifth aspect of the invention relates to an isolated peptide havingthe amino acid sequence of:

(i) KPXDSXSXJAKJJSXJJXSJJX (SEQ ID NO:5, modified P15b/P20 consensus),wherein

-   -   X at position 3 is N, D, or isoD;    -   X at position 6 is Q, E, g-glutamate, G, A, or S;    -   X at position 8 is N, D, or isoD;    -   X at position 15 is optional and can be any amino acid;    -   X at position 18 is M, E, g-glutamate, G, A, S, T, or K;    -   X at position 22 is optional and can be Q, E, g-glutamate, G, A,        or S; and        J at positions 9, 12, 13, 16, 17, 20, and 21 are hydrophobic        amino acids selected from L, V, I, and F, except that at least        one of the amino acids at these positions is a non-hydrophobic        amino acid or A;

or

(ii) JAKJJSXJJXSJJX (SEQ ID NO: 6, modified P15/20 min consensus),wherein

-   -   X at position 7 is optional and can be any amino acid;    -   X at position 10 is M, E, g-glutamate, G, A, S, T, or K;    -   X at position 14 is optional and can be Q, E, g-glutamate, G, A,        or S; and        J at positions 1, 4, 5, 8, 9, 12, and 13 are hydrophobic amino        acids selected from L, V, I, and F, except that at least one of        the amino acids at these positions is a non-hydrophobic amino        acid or A; and        wherein the peptide induces an active plant response, but does        not induce a hypersensitive response, when applied to plant        tissue.

A sixth aspect of the invention relates to an isolated peptide havingthe amino acid sequence of:

PSPJTXJJXXJJGXJJXAXN (SEQ ID NO: 7, modified P6/6a consensus), wherein

-   -   X at position 6 is Q, E, g-glutamate, G, A, or S;    -   X at position 9 is M, E, g-glutamate, G, A, S, T, or K;    -   X at position 10 is H or N;    -   X at position 14 is E, g-glutamate, D, or isoD;    -   X at position 17 is Q, E, g-glutamate, G, A, or S;    -   X at position 19 is Q, E, g-glutamate, G, A, or S; and        J at positions 4, 7, 8, 11, 12, 15, and 16 are hydrophobic amino        acids selected from L, V, I, M, and F, except that at least one        of the amino acids at these positions is a non-hydrophobic amino        acid or A; wherein the peptide induces an active plant response,        but does not induce a hypersensitive response, when applied to        plant tissue.

A seventh aspect of the invention relates to an isolated peptide havingthe amino acid sequence of:

(i) XXXXXXJXXJJXXJJXJJK (SEQ ID NO: 8, modified P14d consensus), wherein

-   -   X at position 1 can be: Q, N, D, E, g-glutamate, isoD, or S;    -   X at position 2 can be: D, E, g-glutamate, isoD;    -   X at position 3 can be: P, D, E, isoD, or g-glutamate;    -   X at position 4 can be M, A, S, D, E, isoD, or g-glutamate    -   X at position 5 can be Q, E, or g-glutamate;    -   X at position 6 can be A, E, or g-glutamate;    -   X at position 8 can be M, L, E, Q, D, N, G, A, S, isoD, or        g-glutamate;    -   X at position 9 can be Q, N, E, D, G, A, S, isoD, or        g-glutamate;    -   X at position 12 can be Q, N, E, D, G, A, S, isoD, or        g-glutamate;    -   X at position 13 can be Q, N, E, D, G, A, S, isoD, or        g-glutamate;    -   X at position 16 can be K, Q, N, E, D, R, G, A, or S; and        J at positions 7, 10, 11, 14, 15, 17, and 18 are hydrophobic        amino acids selected from L, V, I, and F, except that at least        one of the amino acids at these positions is a non-hydrophobic        amino acid or A; or        (ii) JXXJJXXJJXJJK (SEQ ID NO: 9, modified P14d min consensus),        wherein    -   X at position 2 can be M, L, E, Q, D, N, G, A, S, isoD, or        g-glutamate;    -   X at position 3 can be Q, N, E, D, G, A, S, isoD, or        g-glutamate;    -   X at position 6 can be Q, N, E, D, G, A, S, isoD, or        g-glutamate;    -   X at position 7 can be Q, N, E, D, G, A, S, isoD, or        g-glutamate;    -   X at position 10 can be K, Q, N, E, D, R, G, A, or S; and        J at positions 1, 4, 5, 8, 9, 11, and 12 are hydrophobic amino        acids selected from L, V, I, and F, except that at least one of        the amino acids at these positions is a non-hydrophobic amino        acid or A;        wherein the peptide induces an active plant response, but does        not induce a hypersensitive response, when applied to plant        tissue.

An eighth aspect of the invention relates to an isolated peptide havingthe amino acid sequence of:

(i) JXXJJXJJXXJJ (SEQ ID NO: 10, modified P25 consensus) wherein

-   -   X at position 2 can be Q, N, E, g-glutamate, D, isoD, T, S, A,        or G;    -   X at position 3 can be K, Q, N, E, g-glutamate, D, isoD, T, S,        A, or G;    -   X at position 6 can be K, Q, N, E, g-glutamate, D, isoD, T, S,        A, or G;    -   X at position 9 can be E, g-glutamate, D, isoD, Q, N, T, S, A,        or G;    -   X at position 10 can be A, G, S, T, E, g-glutamate, D, isoD, Q,        or N; and        J at positions 1, 4, 5, 7, 8, 10, and 11 are hydrophobic amino        acids selected from L, V, I, F, and M, except that at least one        of the amino acids at these positions is a non-hydrophobic amino        acid or A; or        (ii) JXXJJXXJJXJJXXJJ (SEQ ID NO: 11, modified P25 consensus)        wherein    -   X at position 2 can be T, S, A, G, D, isoD, E, g-glutamate, Q,        or N;    -   X at position 3 can be G, T, S, A, D, isoD, E, g-glutamate, Q,        or N;    -   X at position 6 can be Q, N, E, g-glutamate, D, isoD, T, S, A,        or G;    -   X at position 7 can be K, Q, N, E, g-glutamate, D, isoD, T, S,        A, or G;    -   X at position 10 can be K, Q, N, E, g-glutamate, D, isoD, T, S,        A, or G;    -   X at position 13 can be E, g-glutamate, D, isoD, Q, N, T, S, A,        or G;    -   X at position 14 can be A, G, S, T, E, g-glutamate, D, isoD, Q,        or N;        J at positions 1, 4, 5, 8, 9, 11, 12, and 15 are hydrophobic        amino acids selected from L, V, I, F, and M, except that at        least one of the amino acids at these positions is a        non-hydrophobic amino acid or A; and J at position 16 is        optional and a hydrophobic amino acid selected from L, V, I, and        F;        wherein the peptide induces an active plant response, but does        not induce a hypersensitive response, when applied to plant        tissue.

A ninth aspect of the invention relates to an isolated peptidecomprising the amino acid sequence of:

(i) XXXXXXXXXXXJXXJJXXJJXXJJXXX (SEQ ID NO: 12, modified P17/18),wherein

-   -   X at position 1 can be any amino acid, but preferably Q, S, E,        g-glutamate, A, T, G, D, isoD, N, K, or R;    -   X at position 2 can be any amino acid, but preferably Q, S, E,        g-glutamate, A, T, G, D, isoD, N, K, or R;    -   X at position 3 can be any amino acid, but preferably P, Q, S,        E, g-glutamate, A, T, G, D, isoD, N, K, or R;    -   X at position 4 can be any amino acid, but preferably I, Q, S,        E, g-glutamate, A, T, G, D, N, isoD, K, or R;    -   X at position 5 can be any amino acid, but preferably D, isoD,        S, E, g-glutamate, A, T, G, N, Q, K, or R;    -   X at position 6 can be any amino acid, but preferably R, Q, S,        E, g-glutamate, A, T, G, D, isoD, N, or K;    -   X of position 7 can be any amino acid, but preferably Q, S, E,        g-glutamate, A, T, G, D, isoD, N, K, or R;    -   X at position 8 can be any amino acid, but preferably T, Q, S,        E, g-glutamate, A, G, D, isoD, N, K, or R;    -   X at position 9 can be any amino acid, but preferably I, Q, S,        E, g-glutamate, A, T, G, D, isoD, N, K, or R;    -   X at position 10 can be any amino acid, but preferably E,        g-glutamate, Q, S, A, T, G, D, isoD, N, K, or R;    -   X at position 11 can be any amino acid, but preferably Q, S, E,        g-glutamate, A, T, G, D, isoD, N, K, or R;    -   X at position 13 can be any amino acid, but preferably A, S, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 14 can be any amino acid, but preferably Q, A, S,        T, G, D, isoD, E, g-glutamate, N, K, or R;    -   X at position 17 can be any amino acid, but preferably A, S, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 18 can be any amino acid, but preferably Q, A, S,        T, G, D, isoD, E, g-glutamate, N, K, or R;    -   X at position 21 can be any amino acid, but preferably K, A, S,        T, G, D, isoD, E, g-glutamate, Q, N, or R;    -   X at position 22 can be any amino acid, but preferably S, A, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 25 can be any amino acid, but preferably S, A, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 26 can be any amino acid, but preferably P, S, A,        T, G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 27 can be any amino acid, but preferably Q, S, A,        T, G, D, isoD, E, g-glutamate, N, K, or R;        J at positions 12, 15, 16, 19, 20, 23, and 24 are hydrophobic        amino acids selected from L, V, I, F, and M, except that at        least one of the amino acids at these positions is a        non-hydrophobic amino acid or A; or        (ii) JXXJJXXJJXXJJ (SEQ ID NO: 13, modified P17/18 min        consensus), wherein    -   X at position 2 can be any amino acid, but preferably A, S, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 3 can be any amino acid, but preferably Q, A, S,        T, G, D, isoD, E, g-glutamate, N, K, or R;    -   X at position 6 can be any amino acid, but preferably A, S, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 7 can be any amino acid, but preferably Q, A, S,        T, G, D, isoD, E, g-glutamate, N, K, or R;    -   X at position 10 can be any amino acid, but preferably K, A, S,        T, G, D, isoD, E, g-glutamate, Q, N, or R;    -   X at position 11 can be any amino acid, but preferably S, A, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R; and        J at positions 1, 4, 5, 8, 9, 12, and 13 are hydrophobic amino        acids selected from L, V, I, F, and M, except that at least one        of the amino acids at these positions is a non-hydrophobic amino        acid or A;        wherein the peptide induces an active plant response, but does        not induce a hypersensitive response, when applied to plant        tissue.

A tenth aspect of the invention relates to an isolated peptidecomprising the amino acid sequence of:

XJXXJJXJJXXJJ (SEQ ID NO: 14, modified P19 consensus), wherein

-   -   X at position 1 is optional and can be L, I, V, F, or M;    -   X at position 3 can be any amino acid, but preferably K, A, S,        T, G, D, isoD, E, g-glutamate, Q, N, or R;    -   X at position 4 can be any amino acid, but preferably A, S, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 7 can be any amino acid, but preferably K, A, S,        T, G, D, isoD, E, g-glutamate, Q, N, or R;    -   X at position 10 can be any amino acid, but preferably A, S, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 11 can be any amino acid, but preferably R, A, S,        T, G, D, isoD, E, g-glutamate, Q, N, or K; and        J at positions 2, 5, 6, 8, 9, 12, and 13 are hydrophobic amino        acids selected from L, V, I, F, and M, except that at least one        of the amino acids at these positions is a non-hydrophobic amino        acid or A;        wherein the peptide induces an active plant response, but does        not induce a hypersensitive response, when applied to plant        tissue.

An eleventh aspect of the invention relates to an isolated peptide thatincludes the amino acid sequence of:

Z₁-LLXLFXXIL-Z₂ (SEQ ID NO: 126, P3 minimum) wherein

-   -   X at position 3 is any hydrophilic amino acid, preferably K, A,        S, T, G, D, isoD, E, g-glutamate, Q, N, or R;    -   X at position 6 is any hydrophilic amino acid, preferably K, A,        S, T, G, D, isoD, E, g-glutamate, Q, N, or R;    -   X at position 7 is any hydrophilic amino acid, preferably K, A,        S, T, G, D, isoD, E, g-glutamate, Q, N, or R; and    -   wherein one of Z₁ and Z₂ is present, but preferably not both,        with Z₁ comprising LXX- where each X is a hydrophilic amino        acid, preferably K, A, S, T, G, D, isoD, E, g-glutamate, Q, N,        or R, and with Z₂ comprising XXLF where each X is a hydrophilic        amino acid, preferably K, A, S, T, G, D, isoD, E, g-glutamate,        Q, N, or R.

A twelfth aspect of the invention relates to an isolated peptide thatincludes the amino acid sequence of:L-X-X-(L/I)-(L/I)-X-X-(L/I/V)-(L/I/V) (SEQ ID NO: 116), wherein thepeptide is free of cysteine and methionine; each X at positions 2, 3, 6,7 is any amino acid; and the peptide induces an active plant response,but does not induce a hypersensitive response, when applied to planttissue.

A thirteenth aspect of the invention relates to a fusion protein thatincludes one of the peptides of the first through twelfth aspects of theinvention along with one or more of a purification tag, a solubilitytag, or a second peptide according to one of the first through twelfthaspects of the invention.

A fourteenth aspect of the invention relates to an isolated peptideselected from the group consisting of DVGQLIGELIDRGLQ (SEQ ID NO: 15),GDVGQLIGELIDRGLQSVLAG (SEQ ID NO: 16), SSRALQEVIAQLAQELTHN (SEQ ID NO:17), or GLEDIKAALDTLIHEKLG (SEQ ID NO: 18). Also encompassed by thisaspect of the invention are fusion proteins that include one of thesepeptides along with one or more of a purification tag, a solubility tag,or a second peptide according to one of the first through twelfth andfourteenth aspects of the invention.

A fifteenth aspect of the invention relates to a composition thatincludes one or more peptides or fusion proteins according to the first,second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth,eleventh, twelfth, thirteenth, or fourteenth aspects of the invention,and a carrier.

A sixteenth aspect of the invention relates to a method of impartingdisease resistance to plants. This method includes: applying aneffective amount of an isolated peptide or fusion protein according tothe first, second, third, fourth, fifth, sixth, seventh, eighth, ninth,tenth, eleventh, twelfth, thirteenth or fourteenth aspects of theinvention or a composition according to the fifteenth aspect of theinvention to a plant or plant seed or the locus where the plant isgrowing or is expected to grow, wherein said applying is effective toimpart disease resistance.

A seventeenth aspect of the invention relates to a method of enhancingplant growth. This method includes: applying an effective amount of anisolated peptide or fusion protein according to the first, second,third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh,twelfth, thirteenth or fourteenth aspects of the invention or acomposition according to the fifteenth aspect of the invention to aplant or plant seed or the locus where the plant is growing or isexpected to grow, wherein said applying is effective to enhance plantgrowth.

An eighteenth aspect of the invention relates to a method of increasinga plant's tolerance and resistance to biotic stressors. This methodincludes: applying an effective amount of an isolated peptide or fusionprotein according to the first, second, third, fourth, fifth, sixth,seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth orfourteenth aspects of the invention or a composition according to thefifteenth aspect of the invention to a plant or plant seed or the locuswhere the plant is growing or is expected to grow, wherein said applyingis effective to increase the plant's tolerance and resistance to bioticstress factors selected from the group consisting of pests such asinsects, arachnids, nematodes, weeds, and combinations thereof.

A nineteenth aspect of the invention relates to a method of increasing aplant's tolerance to abiotic stress. This method includes: applying aneffective amount of an isolated peptide or fusion protein according tothe first, second, third, fourth, fifth, sixth, seventh, eighth, ninth,tenth, eleventh, twelfth, thirteenth or fourteenth aspects of theinvention or a composition according to the fifteenth aspect of theinvention to a plant or plant seed or the locus where the plant isgrowing or is expected to grow, wherein said applying is effective toincrease the plant's tolerance to abiotic stress factors selected fromthe group consisting of salt stress, water stress (including drought andflooding), ozone stress, heavy metal stress, cold stress, heat stress,nutritional stress (phosphate, potassium, nitrogen deficiency) andcombinations thereof.

A twentieth aspect of the invention relates to a method impartingdesiccation resistance to cuttings removed from ornamental plants. Thismethod includes: applying an isolated peptide or fusion proteinaccording to the first, second, third, fourth, fifth, sixth, seventh,eighth, ninth, tenth, eleventh, twelfth, thirteenth or fourteenthaspects of the invention or a composition according to the fifteenthaspect of the invention to a plant or the locus where the plant isgrowing, wherein said applying is effective to impart desiccationresistance to cuttings removed from the ornamental plant.

A twenty-first aspect of the invention relates to a method of impartingpost-harvest disease or post-harvest desiccation resistance to a fruitor vegetable. This method includes: applying an effective amount of anisolated peptide or fusion protein according to the first, second,third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh,twelfth, thirteenth or fourteenth aspects of the invention or acomposition according to the fifteenth aspect of the invention to aplant containing a fruit or vegetable or the locus where the plant isgrowing; or applying an effective amount of the isolated peptide or thecomposition to a harvested fruit or vegetable, wherein said applying iseffective to impart post-harvest disease resistance or desiccationresistance to the fruit or vegetable.

A twenty-second aspect of the invention relates to a method of enhancingthe longevity of fruit or vegetable ripeness. This method includes:applying an effective amount of an isolated peptide or fusion proteinaccording to the first, second, third, fourth, fifth, sixth, seventh,eighth, ninth, tenth, eleventh, twelfth, thirteenth or fourteenthaspects of the invention or a composition according to the fifteenthaspect of the invention to a plant containing a fruit or vegetable orthe locus where the plant is growing; or applying an effective amount ofthe isolated peptide or the composition to a harvested fruit orvegetable, wherein said applying is effective to enhance the longevityof fruit or vegetable ripeness.

A twenty-third aspect of the invention relates to a method of modulatingone or more biological signaling processes of a plant. This methodincludes: applying an effective amount of an isolated peptide accordingto the first, second, third, fourth, fifth, sixth, seventh, eighth,ninth, tenth, eleventh, twelfth, thirteenth or fourteenth aspects of theinvention or a composition according to the fifteenth aspect of theinvention to a plant or the locus where the plant is growing, whereinsaid applying is effective in initiating one or more biochemicalsignaling processes.

A twenty-fourth aspect of the invention relates to a DNA constructincluding a first nucleic acid molecule encoding a peptide according tothe first, second, third, fourth, fifth, sixth, seventh, eighth, ninth,tenth, eleventh, twelfth, thirteenth or fourteenth aspects of theinvention or a fusion polypeptide containing the same; and apromoter-effective nucleic acid molecule operably coupled to the firstnucleic acid molecule. This aspect of the invention also encompasses arecombinant expression vector containing the DNA construct, arecombinant host cell containing the DNA construct, as well astransgenic plants or plant seeds that include a recombinant plant cellof the invention (which contains the DNA construct).

A twenty-fifth aspect of the invention relates to a method of impartingdisease resistance to plants, enhancing plant growth, impartingtolerance and resistance to biotic stressors, imparting tolerance toabiotic stress, or modulating plant biochemical signaling. This methodincludes providing a transgenic plant transformed with a DNA constructaccording to the twenty-fourth aspect of the invention; and growing theplant under conditions effective to permit the DNA construct to expressthe peptide or the fusion polypeptide to impart disease resistance,enhance plant growth, impart tolerance and resistance to bioticstressors, impart tolerance to abiotic stress, or modulate biochemicalsignaling to the transgenic plant.

A twenty-sixth aspect of the invention relates to a method of impartingdesiccation resistance to cuttings removed from ornamental plants,imparting post-harvest disease or post-harvest desiccation resistance toa fruit or vegetable, or enhancing the longevity of fruit or vegetableripeness. The method includes providing a transgenic plant transformedwith a DNA construct according to the twenty-fourth aspect of theinvention; and growing the plant under conditions effective to permitthe DNA construct to express the peptide or the fusion polypeptide toimpart desiccation resistance to cuttings removed from a transgenicornamental plant, impart post-harvest disease resistance or desiccationresistance to a fruit or vegetable removed from the transgenic plant, orenhance longevity of ripeness for a fruit or vegetable removed from thetransgenic plant.

A twenty-seventh aspect of the invention relates to a method ofimparting disease resistance to plants, enhancing plant growth,imparting tolerance and resistance to biotic stressors, impartingtolerance to abiotic stress, or modulating biochemical signaling. Thismethod includes providing a transgenic plant seed transformed with a DNAconstruct according to the twenty-fourth aspect of the invention;planting the transgenic plant seed in soil; and propagating a transgenicplant from the transgenic plant seed to permit the DNA construct toexpress the peptide or the fusion polypeptide to impart diseaseresistance, enhance plant growth, impart tolerance to biotic stress andresistance to biotic stressors, or impart tolerance to abiotic stress.

A twenty-eighth aspect of the invention relates to a method of impartingdesiccation resistance to cuttings removed from ornamental plants,imparting post-harvest disease or post-harvest desiccation resistance toa fruit or vegetable, or enhancing the longevity of fruit or vegetableripeness. The method includes providing a transgenic plant seedtransformed with a DNA construct according to the twenty-fourth aspectof the invention; planting the transgenic plant seed in soil; andpropagating a transgenic plant from the transgenic plant seed to permitthe DNA construct to express the peptide or the fusion polypeptide toimpart desiccation resistance to cuttings removed from a transgenicornamental plant, impart post-harvest disease resistance or desiccationresistance to a fruit or vegetable removed from the transgenic plant, orenhance longevity of ripeness for a fruit or vegetable removed from thetransgenic plant.

By providing peptides that do not elicit a hypersensitive response butelicit other active plant responses, including, among others, peroxideproduction, growth enhancement, and resistance to biotic and abioticstress, where such peptides desirably exhibit improved solubility,stability, resistance to chemical degradation, or a combination of theseproperties, it will afford growers with greater flexibility inpreparing, handling, and delivering to plants in their fields orgreenhouses effective amounts of compositions containing theseHR-negative peptides. Simplifying the application process for growerswill lead to greater compliance and, thus, improved results with respectto one or more of disease resistance, growth enhancement, tolerance andresistance to biotic stressors, tolerance to abiotic stress, desiccationresistance for cuttings removed from ornamental plants, post-harvestdisease resistance or desiccation resistance to fruit or vegetablesharvested from plants, and/or improved longevity of fruit or vegetableripeness for fruit or vegetables harvested from plants. These and otherbenefits are described herein.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention relates to novel peptides that possess theability to induce an active plant response, but not a hypersensitiveresponse, that afford one or more of the following attributes: modifiedbiochemical signaling; enhanced growth; pathogen resistance; and/orbiotic or abiotic stress resistance.

As used herein, naturally occurring amino acids are identifiedthroughout by the conventional three-letter and/or one-letterabbreviations, corresponding to the trivial name of the amino acid, inaccordance with the following list: Alanine (Ala, A), Arginine (Arg, R),Asparagine (Asn, N), Aspartic acid (Asp, D), Cysteine (Cys, C), Glutamicacid (Glu, E), Glutamine (Gln, Q), Glycine (Gly, G), Histidine (His, H),Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met,M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine(Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V).The abbreviations are accepted in the peptide art and are recommended bythe IUPAC-IUB commission in biochemical nomenclature. Naturallyoccurring variations of amino acids include, without limitation,gamma-glutamate (g-Glu) and isoaspartate (iso-Asp or isoD).

The term “amino acid” further includes analogues, derivatives, andcongeners of any specific amino acid referred to herein, as well asC-terminal or N-terminal protected amino acid derivatives (e.g.,modified with an N-terminal, side chain, or C-terminal protecting group,including but not limited to acetylation, formylation, methylation,amidation, esterification, PEGylation, and addition of lipids).Non-naturally occurring amino acids are well known and can be introducedinto peptides of the present invention using solid phase synthesis asdescribed below. Furthermore, the term “amino acid” includes both D- andL-amino acids. Hence, an amino acid which is identified herein by itsname, three letter or one letter symbol and is not identifiedspecifically as having the D or L configuration, is understood to assumeany one of the D or L configurations. In one embodiment, a peptidecomprises all L-amino acids.

In certain embodiments, peptides are identified to “consist of” arecited sequence, in which case the peptide includes only the recitedamino acid sequence(s) without any extraneous amino acids at the N- orC-terminal ends thereof. To the extent that a recited sequence is in theform of a consensus sequence where one or more of the denoted X or Xaaresidues can be any of one or more amino acids, then multiple peptidesequences are embraced by a peptide consisting of such a recitedsequence.

In certain other embodiments, peptides are identified to “consistessentially of” a recited sequence, in which case the peptide includesthe recited amino acid sequence(s) optionally with one or moreextraneous amino acids at the N- and/or C-terminal ends thereof, whichextraneous amino acids do not materially alter one or more of thefollowing properties: (i) the ability of the peptide to induce an activeplant response, (ii) solubility of the peptide in water or aqueoussolutions, (iii) stability of the peptide dissolved in water or aqueoussolution at 50° C. over a period of time (e.g., 3 weeks), (iv)resistance of the peptide to chemical degradation in the presence of anaqueous buffered solution that includes a biocidal agent (e.g.,Proxel®GXL) at 50° C. over a period of time (e.g., 3 weeks); and (v) theinability of the peptide to induce a hypersensitive response uponinfiltration or application to plants.

Briefly, the stability and resistance to chemical degradation ofpeptides can be assessed as follows using peptide samples having aninitial purity of at least about 80%, at least about 82%, at least about84%, at least about 86%, at least about 88%, at least about 90%, atleast about 92%, at least about 94%, at least about 96%, or at leastabout 98%. For water stability, the peptide is dissolved directly inde-ionized water. For chemical degradation tests, the peptide isdissolved in an aqueous solution containing 50 mM pH buffer and 0.25%Proxel GXL. Exemplary pH buffers include, without limitation: (i)Citrate pH 5.6; (ii) MES pH 6.2; (iii) MOPS pH 6.5; (iv) imidazole pH7.0; (v) Citrate pH 7.2; (vi) EDDS, pH 7.3; (vii) EDTA pH 8.0; (viii)sodium phosphate pH 8.0; or (ix) TES pH 8.0. Peptides are firstdissolved in the aqueous solution at a concentration of 0.5 mg/ml. Thesamples are incubated at 50° C. to allow for accelerated degradation. Aninitial sample of the peptide is removed, diluted 10× with water, andanalyzed by reverse-phase HPLC. Briefly, 20 μl of the sample is injectedinto the solvent flow of an HPLC instrument and analyzed on a C18 HPLCcolumn (YMC ProPack C18, YMC, Japan, or C18 Stablebond, AgilentTechnologies, USA) using either a triethylamine phosphate inwater/acetonitrile gradient or a 0.1% TFA in water/0.1% TFA inacetonitrile gradient to separate different peptide species. Elutingpeptides are monitored by UV absorbance at 218 nm and quantified basedon the area under the peak. The area under the peak for the initialpeptide sample is treated as the standard for relative quantification insubsequent runs. At regular intervals (e.g., 1, 3, 7, 10, 14, 17, and 21days), each peptide sample is surveyed and analyzed by HPLC as describedabove. If necessary to observe degradation (i.e., where the peptideexhibits a high degree of chemical stability), this protocol can beextended by several weeks to observe degradation. The quantification ofsubsequent peptide runs is expressed as a percentage of the original(day 0) HPLC result.

A peptide that is at least partially soluble in water or aqueoussolution exhibits a solubility of greater than 0.1 mg/ml, preferably atleast about 1.0 mg/ml, at least about 2.0 mg/ml, at least about 3.0mg/ml, or at least about 4.0 mg/ml. In certain embodiments, the peptideexhibits high solubility in water or aqueous solution, with a solubilityof at least about 5.0 mg/ml, at least about 10.0 mg/ml, at least about15.0 mg/ml, or at least about 20 mg/ml.

A peptide that is stable in water or aqueous solution exhibits at leastabout 66%, at least about 68%, at least about 70%, at least about 72%,at least about 74%, at least about 76%, at least about 78%, at leastabout 80%, at least about 82%, at least about 84%, at least about 86%,at least about 88%, or at least about 90% of the original peptideconcentration over the designated period of time incubated at 50° C. Incertain embodiments, the designated period of time is 3 days, 7 days, 14days, 21 days, 28 days, one month, two months, or three months.

A peptide that is resistant to chemical degradation exhibits at leastabout 66%, at least about 68%, at least about 70%, at least about 72%,at least about 74%, at least about 76%, at least about 78%, at leastabout 80%, at least about 82%, at least about 84%, at least about 86%,at least about 88%, or at least about 90% of the original peptideconcentration over the designated period of time incubated at 50° C. Incertain embodiments, the designated period of time is 3 days, 7 days, 14days, 21 days, 28 days, one month, two months, three months, or fourmonths. Four months of stability at 50° C. is roughly equivalent to 2years of stability at room temperature.

A property of a peptide to elicit a hypersensitive response, or not,upon infiltration or application of the peptide to plant tissues can bemeasured by applying the peptide in dry powder form or in solution formto a plant, particularly though not exclusively a plant leaf.Application rates include 1-500 ug/ml for liquid solution and0.0001-0.5% (w/w for powder application. Exemplary application of thepeptide in solution form is described in the accompanying Examples.Plants are considered HR-positive (“HR+”) if they exhibit wide-spreadmacroscopic cell death visible to the naked eye, accompanied by wiltingand browning of the affected tissue within 48 hours. Plants areconsidered HR-negative (“HR−”) if they exhibit no discernible wilting ortissue death observable by naked eye. It is possible that an HR− peptidecould cause the death of a small proportion of cells in treated tissuewhich is not observable by naked eye.

In certain embodiments, material alteration of the one or moreproperties is intended to mean that there is less than 20% variation,less than 15% variation, less than 10% variation, or less than 5%variation in a recited property when comparing a peptide possessing theone or more extraneous amino acids to an otherwise identical peptidelacking the one or more extraneous amino acids. In certain embodiments,the number of extraneous amino acids at the N- or C-terminal ends is upto 20 amino acids at one or both ends, up to 15 amino acids at one orboth ends, up to 10 amino acids at one or both ends, up to 7 amino acidsat one or both ends, up to 5 amino acids at one or both ends, or up to 3amino acids at one or both ends. Further, to the extent that a recitedsequence is in the form of a consensus sequence where one or more of thedenoted X or Xaa residues can be any of one or more amino acids, thenmultiple peptide sequences are embraced by the peptide consistingessentially of such a recited sequence, without regard to additionalvariations of such sequences that are afforded by the presence ofextraneous amino acids at the N- and/or C-terminal ends thereof.

In various embodiments of the invention, the disclosed peptides mayinclude a hydrophilic amino acid sequence, e.g., at either theN-terminal or C-terminal end of a designated peptide sequence. Thehydrophilic amino acid sequence is at least 3, at least 4, at least 5,at least 6, at least 7, at least 8, at least 9, or at least 10 aminoacids in length, and includes amino acid residues that contribute to ahydrophilic property of the amino acid sequence that is adjacent to theamino acid sequence of the designated peptide (i.e., the peptide thatinduces an active plant response). Different methods have been used inthe art to calculate the relative hydrophobicity/hydrophilicity of aminoacid residues and proteins (Kyte et al., “A Simple Method for Displayingthe Hydropathic Character of a Protein,” J. Mol. Biol. 157: 105-32(1982); Eisenberg D, “Three-dimensional Structure of Membrane andSurface Proteins,” Ann. Rev. Biochem. 53: 595-623 (1984); Rose et al.,“Hydrogen Bonding, Hydrophobicity, Packing, and Protein Folding,” Annu.Rev. Biomol. Struct. 22: 381-415 (1993); Kauzmann, “Some Factors in theInterpretation of Protein Denaturation,” Adv. Protein Chem. 14: 1-63(1959), which are hereby incorporated by reference in their entirety).Any one of these hydrophobicity scales can be used for the purposes ofthe present invention; however, the Kyte-Doolittle hydrophobicity scaleis perhaps the most often referenced scale. These hydropathy scalesprovide a ranking list for the relative hydrophobicity of amino acidresidues. For example, amino acids that contribute to hydrophilicityinclude Arg (R), Lys (K), Asp (D), Glu (E), Gln (Q), Asn (N), and His(H) as well as, albeit to a lesser extent, Ser (S), Thr (T), Gly (G),Pro (P), Tyr (Y), and Trp (W). For example, polyglutamate sequences canbe used to enhance solubility of proteins and other drug molecules(Lilie et al, Biological Chemistry 394(8):995-1004 (2013); Li et al.,Cancer Research 58: 2404-2409 (1998)), each of which is herebyincorporated by reference in its entirety).

The “hydropathy index” of a protein or amino acid sequence is a numberrepresenting its average hydrophilic or hydrophobic properties. Anegative hydropathy index defines the hydrophilicity of the amino acidsequence of interest. The hydropathy index is directly proportional tothe hydrophilicity of the amino acid sequence of interest; thus, themore negative the index, the greater its hydrophilicity. In certainembodiments, the added hydrophilic amino acid sequence described abovehas a hydropathy index of less than 0, −0.4, −0.9, −1.3, −1.6, −3.5,−3.9, or −4.5. In certain embodiments, the resulting entire peptide willhave a hydropathy index of less than 0.3, 0.2, 0.1, or 0.0, preferablyless than −0.1, −0.2, −0.3, −0.4, more preferably less than −0.5, −0.6,−0.7, −0.8, −0.9, or −1.0.

In the peptides of the present invention, amino acids that contribute toa hydrophilic hydropathy index, for either the peptide as a whole or theadded hydrophilic amino acid sequence, include Arg (R), Lys (K), Asp(D), Glu (E), Gln (Q), Asn (N), His (H), Ser (S), Thr (T), Gly (G), Pro(P), Tyr (Y), and Trp (W). Of these, Asp (D), Glu (E), Gln (Q), Asn (N)or their variants are preferred. Exemplary variants include g-glutamatefor Glu and isoaspartic acid (or isoD) for Asp.

As used herein, in this and in other aspects of the invention, the term“hydrophobic amino acid” is intended to refer to an amino acid thatcontributes hydrophobicity to the hydropathy index of a designated aminoacid sequence. Amino acids that contribute to a hydrophobic hydropathyindex, for either the peptide as a whole or a particular amino acidsequence thereof, include Ile (I), Val (V), Leu (L), Phe (F), Cys (C),Met (M), and Ala (A). In certain embodiments, the term “hydrophobicamino acid” may refer to any one of Ile (I), Val (V), Leu (L), Phe (F),Cys (C), Met (M), and Ala (A); or, alternatively, to any one of Ile (I),Val (V), Leu (L), Phe (F), and Ala (A). In certain other embodiments,the term “hydrophobic amino acid” may refer to one of Ile (I), Val (V),Leu (L), and Phe (F).

As used herein, the term “non-hydrophobic amino acid” is intended tomean an amino acid that is hydrophilic (or not hydrophobic) on one ofthe above-identified hydrophobicity scales. This term generally refersto those amino acids that contribute to a hydrophilic hydropathy indexfor either the peptide as a whole or the added hydrophilic amino acidsequence.

In one aspect of the invention, the peptide includes the amino acidsequence of:

J-X-X-X-J-J-X-X-X-J-J-X-X-X-J-J (SEQ ID NO: 1)

wherein the peptide is free of cysteine and methionine, each X atpositions 2, 3, 7, 8, 12, and 13 is optional and, when present, is anyamino acid, each X residue at positions 4, 9, and 14 is any amino acid,one to three of the J residues at positions 1, 5, 6, 10, 11, 15, and 16is a non-hydrophobic amino acid or A (preferably, when A is present, itis at one of positions 1, 5, 6, 10, 15, or 16), and all other of the Jresidues are L, I, V, or F, (preferably L, I, or V); and wherein thepeptide induces an active plant response, but does not induce ahypersensitive response, when applied to plant tissue.

In certain embodiments, the peptide does not comprise or consist of theamino acid sequence DVGQLIGELIDRGLQ (SEQ ID NO: 15),GDVGQLIGELIDRGLQSVLAG (SEQ ID NO: 16), SSRALQEVIAQLAQELTHN (SEQ ID NO:17), or GLEDIKAALDTLIHEKLG (SEQ ID NO: 18). Each of these peptides isidentified in Haapalainen et al., “Functional Mapping of Harpin HrpZ ofPseudomonas syringae Reveals the Sites Responsible for ProteinOligomerization, Lipid Interactions, and Plant Defence Induction,” Mol.Plant Pathol. 12(2):151-66 (2011), which is hereby incorporated byreference in its entirety.

In one embodiment, one to three of the J residues at positions 1, 5, 6,10, 11, 15, and 16 is a non-hydrophobic amino acid, and all other arepreferably L, I, V, or F. According to a particular embodiment, not morethan two of the J residues at positions 1, 5, 6, 10, 11, 15, and 16 arenon-hydrophobic. In another embodiment, not more than one of the Jresidues at positions 1, 5, 6, 10, 11, 15, and 16 is non-hydrophobic.

In one embodiment, not more than one of the J residues is A, andpreferably the A residue is at one of positions 1, 5, 6, 10, 15, and 16;one or two of the remaining J residues is optionally a non-hydrophobicamino acid, and all other J residues are L, I, V, or F. In certainembodiments, not more than one of the J residues is A, and preferablythe A residue is at one of positions 1, 5, 6, 10, 15, and 16; and allother J residues are L, I, V, or F.

In certain embodiments, the number of amino acids separating theresidues at position 1 from positions 5 and 6, from positions 10 and 11,and from positions 15 and 16 can vary from one to three amino acids. Inone embodiment, one of the X residues at positions 2 and 3 is notpresent, one of the X residues at positions 7 and 8 is not present, oneof the X residues at positions 12 and 13 is not present, or two or moreof the X residues at these positions is not present. For example, in oneembodiment, one of the X residues at positions 2 and 3 and one of the Xresidues at positions 7 and 8 are not present. In another embodiment,the X residue at position 12, 13, or both, is not present. In anotherembodiment, one of the X residues at positions 2 and 3, one of the Xresidues at positions 7 and 8, and one of the X residues at positions 12and 13 are not present.

In another embodiment, each of the X residues at positions 2, 3, 7, and8 is present (i.e., the peptide includes three amino acids separatingthe residue at position 1 from the residues at positions 5 and 6, andthe residues at positions 5 and 6 from the residues positions 10 and11). In another embodiment, the X residue at position 12 is present orthe X residues at positions 12 and 13 are present (i.e., the peptideincludes two or three amino acids separating the residues at positions10 and 11 from the residues at positions 15 and 16).

In the preceding paragraphs, the residue numbering refers to SEQ ID NO:1, although because certain residues are optional, the actual positionof J residues and intervening residues may differ in any one particularpeptide.

The peptide length in this embodiment is less than 100 amino acids, oralternatively less than 90 amino acids, less than 80 amino acids, lessthan 70 amino acids, less than 60 amino acids, or less than about 50amino acids. In certain embodiments, the peptide length is between 12and about 50 amino acids in length.

In the embodiments described above, where X residues at each ofpositions 2, 3, 7, 8, 12, (when present) of SEQ ID NO: 1 can be anyamino acid, in certain embodiments these residues are independentlyselected from the group of Arg (R), Lys (K), Asp (D), Glu (E), Gln (Q),Asn (N), Ser (S), Thr (T), Gly (G), isoaspartic acid (isoD), andg-glutamate, and each X residue at positions 4, 9, and 13 isindependently selected from the group consisting of G, A, S, T, D, isoD,E, g-glutamate, Q, N, K, and R.

In this embodiment, the isolated peptide is preferably stable whendissolved in water; resistant to chemical degradation in aqueousconditions in the presence of a pH buffer and a biocide, as describedabove; and/or has a solubility in an aqueous solution of at least about1.0 mg/ml.

Exemplary peptides according to the first aspect of the inventioncomprise amino acid sequences identified in Table 1 below:

TABLE 1 Name Sequence SEQ ID NO: P14-22L,26EQAGPQSANKTGNVDDANNQDPLQALEQLLEDLV 19 P14-22A,26LQAGPQSANKTGNVDDANNQDPAQALLQLLEDLV 20 P14-22A,26EQAGPQSANKTGNVDDANNQDPAQALEQLLEDLV 21 P14-22E,26LQAGPQSANKTGNVDDANNQDPEQALLQLLEDLV 22 P14-22E,26EQAGPQSANKTGNVDDANNQDPEQALEQLLEDLV 23 P14-22E,26E-RQAGPQSANEIGNVDDANNQDPEQALEQLLEDLVR 24 P14-22E,26AQAGPQSANKTGNVDDANNQDPE_ALAQLLEDLV 25 P14-22L,26AQAGPQSANKTGNVDDANNQDPLQALAQLLEDLV 26 P14-22A,26AQAGPQSANKTGNVDDANNQDPAQALAQLLEDLV 27 P14-22L,25-26AQAGPQSANKTGNVDDANNQDPLQAAAQLLEDLV 28 P14-22L,28-29AQAGPQSANKTGNVDDANNQDPLQALLQAAEDLV 29 P14-22L,32-33AQAGPQSANKTGNVDDANNQDPLQALLQLLEDAA 30 P14-22L,26,29AQAGPQSANKTGNVDDANNQDPLQALAQLAEDLV 31 P14-22L,29,32AQAGPQSANKTGNVDDANNQDPLQALLQLAEDAV 32 P14f-7D                  QDPAQADEQLLEDLVKLLK 33 P14f-10D                  QDPAQALEQDLEDLVKLLK 34 P14f-11D                  QDPAQALEQLDEDLVKLLK 35 P14f-14D                  QDPAQALEQLLEDDVKLLK 36 P14f-15D                  QDPAQALEQLLEDLDKLLK 37 P14f-17D                  QDPAQALEQLLEDLVKDLK 38 P14f-18D                  QDPAQALEQLLEDLVKLDK 39 P14f-7S                  QDPAQASEQLLEDLVKLLK 40 P14f-10S                  QDPAQALEQSLEDLVKLLK 41 P14f-11S                  QDPAQALEQLSEDLVKLLK 42 P14f-14S                  QDPAQALEQLLEDSVKLLK 43 P14f-15S                  QDPAQALEQLLEDLSKLLK 44 P14f-17S                  QDPAQALEQLLEDLVKSLK 45 P14f-18S                  QDPAQALEQLLEDLVKLSK 46

In certain embodiments, these peptides consist essentially of, orconsist of, the amino sequence of one or more of SEQ ID NOS: 19-46.

Other exemplary peptides according to the first aspect of the inventioncomprise the amino acid sequences identified in Table 2 below:

TABLE 2 SEQ ID Name Sequence NO: P4-14S-13A SQGISEKQLDQLASQLIQALLQP 47P4-14S-13D SQGISEKQLDQLDSQLIQALLQP 48 P4-14S-17D SQGISEKQLDQLLSQLDQALLQP49 P4-14S-21D SQGISEKQLDQLLSQLIQALDQP 50 P4-d18 SQGISEKQLDQLLSQLI_ALLQP51 P4-14S-9S SQGISEKQSDQLLSQLIQALLQP 52 P4-14S-9YSQGISEKQYDQLLSQLIQALLQP 53 P4-14S-13Q SQGISEKQLDQLQSQLIQALLQP 54P4-14S-16S SQGISEKQLDQLLSQSIQALLQP 55 P4-14S-20S SQGISEKQLDQLLSQLIQASLQP56 P4-14S-9A SQGISEKQADQLLSQLIQALLQP 57 P4-14S-9DSQGISEKQDDQLLSQLIQALLQP 58 P4-14S-12D SQGISEKQLDQDLSQLIQALLQP 59P4-14S-16A SQGISEKQLDQLLSQAIQALLQP 60 P4-14S-17S SQGISEKQLDQLLSQLSQALLQP61 P4-d10,14,18 SQGISEKQL_QLL_QLI_ALLQP 62 P4421QSQGISEKQLDQLLSQLIQAQLLQP 63 P4-i21H SQGISEKQLDQLLSQLIQAHLLQP 64 P4410ASQGISEKQLADQLLSQLIQALLQP 65 P4-i14A SQGISEKQLDQLLASQLIQALLQP 66 P4-i18ASQGISEKQLDQLLSQLIAQALLQP 67 P4410A,14A,18A SQGISEKQLADQLLASQLIAQALLQP 68

In certain embodiments, these peptides consist essentially of, orconsist of, the amino sequence of one or more of SEQ ID NOS: 47-68.

Further exemplary peptides according to the first aspect of theinvention comprise the amino acid sequences identified in Table 3 below:

TABLE 3 Name Sequence SEQ ID NO: P25min-7D  SEEEEE DTGVLQKLLKILEAL 69P25min-10D SEEEEELTGDLQKLLKILEAL 70 P25min-11D SEEEEELTGVDQKLLKILEAL 71P25min-14D SEEEEELTGVLQKDLKILEAL 72 P25min-15D SEEEEELTGVLQKLDKILEAL 73P25min-17D SEEEEELTGVLQKLLKDLEAL 74 P25min-18D SEEEEELTGVLQKLLKIDEAL 75P25min-21D SEEEEELTGVLQKLLKILEAD 76 P25min-7S  SEEEEE STGVLQKLLKILEAL 77P25min-10S SEEEEELTGSLQKLLKILEAL 78 P25min-11S SEEEEELTGVSQKLLKILEAL 79P25min-14S SEEEEELTGVLQKSLKILEAL 80 P25min-15S SEEEEELTGVLQKLSKILEAL 81P25min-17S SEEEEELTGVLQKLLKSLEAL 82 P25min-18S SEEEEELTGVLQKLLKISEAL 83P25min-21S SEEEEELTGVLQKLLKILEAS 84In Table 3, the peptides include the solubility tag SEEEEE, indicated byitalic print. Peptides comprising the sequences shown in Table 3 butlacking this specific solubility tag (or having a different solubilitytag) are also contemplated herein.

In certain embodiments, these peptides consist essentially of, orconsist of, the amino sequence of one or more of SEQ ID NOS: 69-84.

Peptides of the first aspect of the invention may also comprise theamino acid sequences identified in Table 4 below:

TABLE 4 Name Sequence SEQ ID NO: P18min-7D  SEEEEE DAQLLAQLLKSLL 85P18min-10D SEEEEELAQDLAQLLKSLL 86 P18min-11D SEEEEELAQLDAQLLKSLL 87P18min-14D SEEEEELAQLLAQDLKSLL 88 P18min-15D SEEEEELAQLLAQLDKSLL 89P18min-18D SEEEEELAQLLAQLLKSDL 90 P18min-19D SEEEEELAQLLAQLLKSLD 91P18min-7S  SEEEEE SAQLLAQLLKSLL 92 P18min-10S SEEEEELAQSLAQLLKSLL 93P18min-11S SEEEEELAQLSAQLLKSLL 94 P18min-14S SEEEEELAQLLAQSLKSLL 95P18min-15S SEEEEELAQLLAQLSKSLL 96 P18min-18S SEEEEELAQLLAQLLKSSL 97P18min-19S SEEEEELAQLLAQLLKSLS 98In Table 4, the peptides include the solubility tag SEEEEE, indicated byitalic print. Peptides comprising the sequences shown in Table 4 butlacking this specific solubility tag (or having a different solubilitytag) are also contemplated herein.

In certain embodiments, these peptides consist essentially of, orconsist of, the amino sequence of one or more of SEQ ID NOS: 85-98.

In another embodiment of the first aspect of the invention, peptidescomprise the amino acid sequences identified in Table 5 below:

TABLE 5 Name Sequence SEQ ID NO: P19min-7D  SEEEEE DKALLKLIARLL  99P19min-10D SEEEEELKADLKLIARLL 100 P19min-11D SEEEEELKALDKLIARLL 101Pl9min-13D SEEEEELKALLKDIARLL 102 P19min-7D  SEEEEE DKALLKLIARLL  99P19min-14D SEEEEELKALLKLDARLL 103 P19min-17D SEEEEELKALLKLIARDL 104P19min-18D SEEEEELKALLKLIARLD 105 P19min-7S  SEEEEE SKALLKLIARLL 106P19min-10S SEEEEELKASLKLIARLL 107 P19min-11S SEEEEELKALSKLIARLL 108P19min-13S SEEEEELKALLKSIARLL 109 P19min-14S SEEEEELKALLKLSARLL 110P19min-17S SEEEEELKALLKLIARSL 111 P19min-18S SEEEEELKALLKLIARLS 112In Table 5, the peptides include the solubility tag SEEEEE, indicated byitalic print. Peptides comprising the sequences shown in Table 5 butlacking this specific solubility tag (or having a different solubilitytag) are also contemplated herein.

In certain embodiments, these peptides consist essentially of, orconsist of, the amino sequence of one or more of SEQ ID NOS: 99-112.

Another aspect of the invention relates to an isolated peptidecomprising the amino acid sequence of one of SEQ ID NOS: 113, 114,117-123, 127, 133-138, 140-142, 144, 145, 148-150, and 153-155, as shownin Table 6 below:

TABLE 6 SEQ Name Sequence ID NO: P14 QAGPQSANKTGNVDDANNQDPMQALMQLLEDLV113 P14a                ANNQDPMQALMQLLEDLV 114 P14-dc1QAGPQSANKTGNVDDANNQDPMQALMQLLEDL 121 P14-dc2QAGPQSANKTGNVDDANNQDPMQALMQLLED 122 P14-dc4QAGPQSANKTGNVDDANNQDPMQALMQLL 123 P14-40                  SEEEEELMQLLEDLV 127 P14-dN2  GPQSANKTGNVDDANNQDPMQALMQLLEDLV 117 P14-dN4    QSANKTGNVDDANNQDPMQALMQLLEDLV 118 P14-dN6      ANKTGNVDDANNQDPMQALMQLLEDLV 119 P14-dN8        KTGNVDDANNQDPMQALMQLLEDLV 120 P4-14S-13V   SQGISEKQLDQLVSQLIQALLQP 133 P4-14S-13F    SQGISEKQLDQLFSQLIQALLQP 134P4-14S-16V    SQGISEKQLDQLLSQVIQALLQP 135 P4-14S-17F   SQGISEKQLDQLLSQLFQALLQP 136 P4-14S-20V    SQGISEKQLDQLLSQLIQAVLQP 137P4-14S-20F    SQGISEKQLDQLLSQLIQAFLQP 138 P4-116    SQGISEKQLDQLLSQL 140P4-117    SQGISEKQLDQLLSQ 141 P18-8 QQPIDRQTIEQMAQLLAQLL 142 P19-9   SEEEEEIGDNPLLKALLKLIA 144 P19-10        SEEEEEELLKALLKLIA 145 P15-62   KPNDSQSNIAKLISALI 148 P15-63     SEEEEEEIAKLISALI 149 P15-60   SEEEEEEEIAKLISALIESLL 150 P25-12      SEEEEELTGVLQKLLK_ILE 153 P25-13   SEEEEELTLTGVLQKLLKILE 154 P25-14    SEEEEELTLTGVLQKLLKIL 155In Table 6, several peptides include the solubility tags S-polyE wherethe polyE segment contains from 3 to 7 Glu (E) residues, indicated byitalic print. Peptides comprising the sequences shown in Table 6 butlacking this specific solubility tag (or having a different solubilitytag) are also contemplated herein.

In certain embodiments, these peptides consist essentially of, orconsist of, the amino sequence of one or more of SEQ ID NOS: 113, 114,117-123, 127, 133-138, 140-142, 144, 145, 148-150, 153-159.

Another aspect of the invention relates to an isolated peptide havingthe amino acid sequence of:

XXGISEKXXXXXXXXXXXXXXXX (SEQ ID NO: 2, modified P1/P4 consensus),wherein

-   X at position 1 is optional and can be S, N, D, isoD, G, A, or S;-   X at position 2 is optional and can be Q, E, g-glutamate, G, A, or    S;-   X at position 8 is Q, E, g-glutamate, G, A, or S;-   X at position 9 is M, L, I, F, or V, or a non-hydrophobic amino    acid;-   X at position 10 is optional and can be D or isoD;-   X at position 11 is Q, E, g-glutamate, G, A, or S;-   X at position 12 is M, L, I, or F, or a non-hydrophobic amino acid;-   X at position 13 is M, L, or I, or a non-hydrophobic amino acid;-   X at position 14 is optional and can be any hydrophilic amino acid,    S, T, D, isoD, K, or Q, and optionally A or C;-   X at position 15 is Q, E, g-glutamate, G, A, S, K, or I;-   X at position 16 is M, L, I, V, or F, or a non-hydrophobic amino    acid;-   X at position 17 is M, L, I, A, or V, or a non-hydrophobic amino    acid;-   X at position 18 is Q, E, g-glutamate, G, A, S, M, T, or K;-   X at position 19 is A, D, isoD, S, V, T, K, R, E, H, or G;-   X at position 20 is M, L, or I;-   X at position 21 is M, L, I, V, S, or F, or a non-hydrophobic amino    acid other than serine;-   X at position 22 is Q, E, g-glutamate, G, A, S;-   X at position 23 is P, Q, E, g-glutamate, G, A, or S; and    wherein at least one of the residues at positions 9, 12, 13, 16, 17,    and 20 is a non-hydrophobic amino acid, or the residue at position    21 is a non-hydrophobic amino acid other than serine; and wherein    the peptide induces an active plant response, but does not induce a    hypersensitive response, when applied to plant tissue.

In this embodiment, the isolated peptide is preferably stable whendissolved in water; resistant to chemical degradation in aqueousconditions in the presence of a pH buffer and a biocide, as describedabove; and/or has a solubility in an aqueous solution of at least about1.0 mg/ml.

One exemplary family of peptides according to this aspect of theinvention have the amino acid sequence of:

SXGISEKXXDXXXXXXXXAXXXP (SEQ ID NO: 3, modified P4 consensus), wherein

X at position 2 is Q, E, g-glutamate, G, A, or S;

X at position 8 is Q, E, g-glutamate, G, A, or S;

X at position 9 is M, S, L, A, I, V, or F, or a non-hydrophobic aminoacid other than serine;

X at position 11 is Q, E, g-glutamate, G, A, or S;

X at position 12 is L, I, or F, or a non-hydrophobic amino acid;

X at position 13 is L, A, I, V, or F, or a non-hydrophobic amino acid;

X at position 14 is any hydrophilic amino acid;

X at position 15 is Q, E, g-glutamate, G, A, S, K, or I;

X at position 16 is L, A, I, V, M, or F, or a non-hydrophobic aminoacid;

X at position 17 is M, I, S, or F, or a non-hydrophobic amino acid otherthan serine;

X at position 18 is Q, E, g-glutamate, G, A, or S;

X at position 20 is M, L, I, V, or F, or a non-hydrophobic amino acid;

X at position 21 is M, L or F, or a non-hydrophobic amino acid; and

X at position 22 is Q, E, g-glutamate, G, A, or S;

wherein one of the residues at positions 9, 12, 13, 16, 20, and 21 is anon-hydrophobic amino acid or A, or the residue at position 17 is anon-hydrophobic amino acid other than serine; and wherein the peptideinduces an active plant response, but does not induce a hypersensitiveresponse, when applied to plant tissue. Preferably, one to four, notmore than three, not more than two, or exactly one of the residues atpositions 9, 12, 13, 16, 20, and 21 is a non-hydrophobic amino acid, orthe residue at position 17 is a non-hydrophobic amino acid other thanserine.

In certain embodiments, these peptides according to SEQ ID NO: 2 alsomeet the structural features defining the peptides of SEQ ID NO: 1, inwhich case methionine and cysteine residues are not present. Thus, inthese embodiments, X at position 14 can be any hydrophilic amino acidother than methionine or cysteine, preferably S or T.

Exemplary peptides that share the consensus structure with SEQ ID NO: 3,or are derived from SEQ ID NO: 3, are identified in Table 7 below:

TABLE 7 Peptide Variants of Peptide P4 (consensus SEQ ID NO: 3) NameSequence SEQ ID NO: P4-14S-13D SQGISEKQLDQLDSQLIQALLQP  48 P4-14S-17DSQGISEKQLDQLLSQLDQALLQP  49 P4-14S-21D SQGISEKQLDQLLSQLIQALDQP  50P4-d18 SQGISEKQLDQLLSQLI_ALLQP  51 P4-14S-9S SQGISEKQSDQLLSQLIQALLQP  52P4-14S-9Y SQGISEKQYDQLLSQLIQALLQP  53 P4-14S-13Q SQGISEKQLDQLQSQLIQALLQP 54 P4-14S-16S SQGISEKQLDQLLSQSIQALLQP  55 P4-14S-20SSQGISEKQLDQLLSQLIQASLQP  56 P4-14S-9D SQGISEKQDDQLLSQLIQALLQP  58P4-14S-12D SQGISEKQLDQDLSQLIQALLQP  59 P4-14S-17SSQGISEKQLDQLLSQLSQALLQP  61 P4-14S-16D SQGISEKQLDQLLSQDIQALLQP 161P4-14S-20D SQGISEKQLDQLLSQLIQADLQP 162 P4-14S-13SSQGISEKQLDQLSSQLIQALLQP 163

In certain other embodiments, the isolated peptide consists essentiallyof, or consists of, the amino acid sequence of one or more of SEQ IDNOS: 48-56, 58, 59, 61 and 161-163.

In this embodiment, the isolated peptide is preferably stable whendissolved in water; resistant to chemical degradation in aqueousconditions in the presence of a pH buffer and a biocide, as describedabove; and/or has a solubility in an aqueous solution of at least about1.0 mg/ml.

Another exemplary family of peptides according to this aspect of theinvention have the amino acid sequence of:

XXGISEKXJDXJJTXJJXAJJXX (SEQ ID NO: 4, modified P1 consensus), wherein

X at position 1 is N, D, isoD, G, A, or S;

X at position 2 is Q, E, g-glutamate, G, A, or S;

X at position 8 is Q, E, g-glutamate, G, A, or S;

X at position 11 is Q, E, g-glutamate, G, A, or S;

X at position 15 is Q, E, g-glutamate, G, A, or S;

X at position 18 is M, T, K, E, g-glutamate, G, A, or S;

X at position 22 is Q, E, g-glutamate, G, A, or S;

X at position 23 is Q, E, g-glutamate, G, A, or S; and

J at positions 9, 12, 13, 16, 17, 20, and 21 are hydrophobic amino acidsselected from L, V, I, and F, except that one of the amino acids atthese positions is a non-hydrophobic amino acid or A;

wherein the peptide induces an active plant response, but does notinduce a hypersensitive response, when applied to plant tissue.Preferably, one to four, not more than three, not more than two, orexactly one of the residues at positions 9, 12, 13, 16, 17, 20, and 21is a non-hydrophobic amino acid or A. In one embodiment, not more thanone of the J residues is A, and preferably the A residue is at one ofpositions 9, 12, 13, 16, 20, and 21; one or two of the remaining Jresidues is optionally a non-hydrophobic amino acid, and all other Jresidues are L, I, V, or F. In certain embodiments, not more than one ofthe J residues is A, and preferably the A residue is at one of positions9, 12, 13, 16, 20, and 21; and all other J residues are L, I, V, or F.

In certain embodiments, these peptides according to SEQ ID NO: 4 alsomeet the structural features defining the peptides of SEQ ID NO: 1, inwhich case methionine and cysteine residues are not present. Thus, inthose embodiments, X at position 18 is T, K, E, g-glutamate, G, A, or S.

In this embodiment, the isolated peptide is preferably stable whendissolved in water; resistant to chemical degradation in aqueousconditions in the presence of a pH buffer and a biocide, as describedabove; and/or has a solubility in an aqueous solution of at least about1.0 mg/ml.

Exemplary peptides that share the consensus structure with SEQ ID NO: 4are identified in Table 8 below:

TABLE 8 Peptide Variants of P1 (consensus SEQ ID NO: 4) Peptide NameSequence SEQ ID NO: P1-13P-20P NQGISEKQLDQLPTQLIMAPLQQ 164 P1NQGISEKQLDQLLTQLIMALLQQ 165 P1-9D NQGISEKQDDQLLTQLIMALLQQ 166 P1-9SNQGISEKQSDQLLTQLIMALLQQ 167 P1-12D NQGISEKQLDQDLTQLIMALLQQ 168 P1-12SNQGISEKQLDQSLTQLIMALLQQ 169 P1-13D NQGISEKQLDQLDTQLIMALLQQ 170 P1-13SNQGISEKQLDQLSTQLIMALLQQ 171 P1-16D NQGISEKQLDQLLTQDIMALLQQ 172 P1-16SNQGISEKQLDQLLTQSIMALLQQ 173 P1-17D NQGISEKQLDQLLTQLDMALLQQ 174 P1-17SNQGISEKQLDQLLTQLSMALLQQ 175 P1-20D NQGISEKQLDQLLTQLIMADLQQ 176 P1-20SNQGISEKQLDQLLTQLIMASLQQ 177 P1-21D NQGISEKQLDQLLTQLIMALDQQ 178 P1-21SNQGISEKQLDQLLTQLIMALSQQ 179

Additional variants of these peptides may include the substitution ofmethionine at position 18 with T, K, E, g-glutamate, G, A, or S, asnoted above.

In certain other embodiments, the isolated peptide consists essentiallyof, or consists of, the amino acid sequence of one or more of SEQ IDNOS: 164, 166-179.

In this embodiment, the isolated peptide is preferably stable whendissolved in water; resistant to chemical degradation in aqueousconditions in the presence of a pH buffer and a biocide, as describedabove; and/or has a solubility in an aqueous solution of at least about1.0 mg/ml.

Yet another aspect of the invention relates to an isolated peptidehaving the amino acid sequence of:

(i) KPXDSXSXJAKJJSXJJXSJJX (SEQ ID NO: 5, modified P15b/P20 consensus),wherein

-   -   X at position 3 is N, D, or isoD;    -   X at position 6 is Q, E, g-glutamate, G, A, or S;    -   X at position 8 is N, D, or isoD;    -   X at position 15 is optional and can be any amino acid;    -   X at position 18 is M, E, g-glutamate, G, A, S, T, or K;    -   X at position 22 is optional and can be Q, E, g-glutamate, G, A,        or S; and    -   J at positions 9, 12, 13, 16, 17, 20, and 21 are hydrophobic        amino acids selected from L, V, I, F, and A, except that at        least one of the amino acids at these positions is a        non-hydrophobic amino acid; or        (ii) JAKJJSXJJXSJJX (SEQ ID NO: 6, modified P15/20 min        consensus), wherein    -   X at position 7 is optional and can be any amino acid;    -   X at position 10 is M, E, g-glutamate, G, A, S, T, or K;    -   X at position 14 is optional and can be Q, E, g-glutamate, G, A,        or S; and    -   J at positions 1, 4, 5, 8, 9, 12, and 13 are hydrophobic amino        acids selected from L, V, I, and F, except that at least one of        the amino acids at these positions is a non-hydrophobic amino        acid or A; and        wherein the peptide induces an active plant response, but does        not induce a hypersensitive response, when applied to plant        tissue. Preferably, one to four, not more than three, not more        than two, or exactly one of the residues at positions 9, 12, 13,        16, 17, 20, and 21 of SEQ ID NO: 5 is a non-hydrophobic amino        acid or A; and one to four, not more than three, not more than        two, or exactly one of the residues at positions 1, 4, 5, 8, 9,        12, and 13 of SEQ ID NO: 6 is a non-hydrophobic amino acid or A.        In one embodiment, not more than one of the J residues is A, and        preferably the A residue is at one of positions 9, 12, 13, 16,        20, and 21 of SEQ ID NO: 5 or positions 1, 4, 5, 8, 12, and 13        of SEQ ID NO: 6; one or two of the remaining J residues is        optionally a non-hydrophobic amino acid, and all other J        residues are L, I, V, or F. In certain embodiments, not more        than one of the J residues is A, and preferably the A residue is        at one of positions 9, 12, 13, 16, 20, and 21 of SEQ ID NO: 5 or        positions 1, 4, 5, 8, 12, and 13 of SEQ ID NO: 6; and all other        J residues are L, I, V, or F.

Exemplary peptides that share the consensus structure with SEQ ID NO:5or 6 are identified in Table 9 below:

TABLE 9 Peptide Variants of P15/20 (consensus SEQ ID NOS: 5 or 6) NameSequence SEQ ID NO: P15b-9D  KPNDSQSNDAKLISALIMSLLQ 180 P15b-12DKPNDSQSNIAKDISALIMSLLQ 181 P15b-13D KPNDSQSNIAKLDSALIMSLLQ 182 P15b-16DKPNDSQSNIAKLISADIMSLLQ 183 P15b-17D KPNDSQSNIAKLISALDMSLLQ 184 P15b-20DKPNDSQSNIAKLISALIMSDLQ 185 P15b-21D KPNDSQSNIAKLISALIMSLDQ 186 P15b-9S KPNDSQSNSAKLISALIMSLLQ 187 P15b-12S KPNDSQSNIAKSISALIMSLLQ 188 P15b-13SKPNDSQSNIAKLSSALIMSLLQ 189 P15b-16S KPNDSQSNIAKLISASIMSLLQ 190 P15b-17SKPNDSQSNIAKLISALSMSLLQ 191 P15b-20S KPNDSQSNIAKLISALIMSSLQ 192 P15b-21SKPNDSQSNIAKLISALIMSLSQ 193

Additional variants of these peptides may include the substitution ofmethionine at position 18 with E, g-glutamate, G, A, S, T, or K, asnoted above.

In certain other embodiments, the peptide consists essentially of, orconsists of, the amino sequence of one or more of SEQ ID NOS: 180-193.

In this embodiment, the isolated peptide is preferably stable whendissolved in water; resistant to chemical degradation in aqueousconditions in the presence of a pH buffer and a biocide, as describedabove; and/or has a solubility in an aqueous solution of at least about1.0 mg/ml.

A further aspect of the invention relates to an isolated peptide havingthe amino acid sequence of:

PSPJTXJJXXJJGXJJXAXN (SEQ ID NO: 7, modified P6/6a consensus), wherein

-   -   X at position 6 is Q, E, g-glutamate, G, A, or S;    -   X at position 9 is M, E, g-glutamate, G, A, S, T, or K;    -   X at position 10 is H or N;    -   X at position 14 is E, g-glutamate, D, or isoD;    -   X at position 17 is Q, E, g-glutamate, G, A, or S;    -   X at position 19 is Q, E, g-glutamate, G, A, or S; and    -   J at positions 4, 7, 8, 11, 12, 15, and 16 are hydrophobic amino        acids selected from L, V, I, M, and F, except that at least one        of the amino acids at these positions in a non-hydrophobic amino        acid or A;        wherein the peptide induces an active plant response, but does        not induce a hypersensitive response, when applied to plant        tissue. Preferably, one to four, not more than three, not more        than two, or exactly one of the residues at positions 4, 7, 8,        11, 12, 15, and 16 is a non-hydrophobic amino acid or A. In one        embodiment, not more than one of the J residues is A, and        preferably the A residue is at one of positions 4, 7, 8, 11, 15,        and 16 of SEQ ID NO: 7; one or two of the remaining J residues        is optionally a non-hydrophobic amino acid, and all other J        residues are L, I, V, M, or F. In certain embodiments, not more        than one of the J residues is A, and preferably the A residue is        at one of positions 4, 7, 8, 11, 15, and 16 of SEQ ID NO: 7; and        all other J residues are L, I, V, M, or F.

Exemplary peptides that share the consensus structure with SEQ ID NO: 7are identified in Table 10 below:

TABLE 10 Peptide Variants of P6/6a (consensus SEQ ID NO: 7) NameSequence SEQ ID NO: P6a-4D  PSPDTQMLMHIVGEILQAQN 194  P6a-7D PSPFTQDLMHIVGEILQAQN 195  P6a-8D  PSPFTQMDMHIVGEILQAQN 196  P6a-11DPSPFTQMLMHDVGEILQAQN 197  P6a-12D PSPFTQMLMHIDGEILQAQN 198  P6a-15DPSPFTQMLMHIVGEDLQAQN 199  P6a-16D PSPFTQMLMHIVGEIDQAQN 200  P6a-4S PSPSTQMLMHIVGEILQAQN 201  P6a-7S  PSPFTQSLMHIVGEILQAQN 202  P6a-8S PSPFTQMSMHIVGEILQAQN 203  P6a-11S PSPFTQMLMHSVGEILQAQN 204  P6a-12SPSPFTQMLMHISGEILQAQN 205  P6a-15S PSPFTQMLMHIVGESLQAQN 206  P6a-16SPSPFTQMLMHIVGEISQAQN 207

Additional variants of these peptides may include the substitution ofmethionine at position 7 with L, I, V, F, a non-hydrophobic amino acid,or A, as noted above; and substitution of methionine at position 9 withL, E, g-glutamate, G, A, S, T, or K, as noted above.

In certain other embodiments, the isolated peptide consists essentiallyof, or consists of, the amino acid sequence of one or more of SEQ IDNOS: 194-207.

In this embodiment, the isolated peptide is preferably stable whendissolved in water; resistant to chemical degradation in aqueousconditions in the presence of a pH buffer and a biocide, as describedabove; and/or has a solubility in an aqueous solution of at least about1.0 mg/ml.

Another aspect of the invention relates to a peptide having the aminoacid sequence of:

(i) XXXXXXJXXJJXXJJXJJK (SEQ ID NO: 8, modified P14d consensus), wherein

-   -   X at position 1 can be: Q, N, D, E, g-glutamate, isoD, or S;    -   X at position 2 can be: D, E, g-glutamate, isoD;    -   X at position 3 can be: P, D, E, isoD, or g-glutamate;    -   X at position 4 can be M, A, S, D, E, isoD, or g-glutamate    -   X at position 5 can be Q, E, or g-glutamate;    -   X at position 6 can be A, E, or g-glutamate;    -   X at position 8 can be M, L, E, Q, D, N, G, A, S, isoD, or        g-glutamate;    -   X at position 9 can be Q, N, E, D, G, A, S, isoD, or        g-glutamate;    -   X at position 12 can be Q, N, E, D, G, A, S, isoD, or        g-glutamate;    -   X at position 13 can be Q, N, E, D, G, A, S, isoD, or        g-glutamate;    -   X at position 16 can be K, Q, N, E, D, R, G, A, or S; and        J at positions 7, 10, 11, 14, 15, 17, and 18 are hydrophobic        amino acids selected from L, V, I, and F, except that at least        one of the amino acids at these positions is a non-hydrophobic        amino acid or A; or        (ii) JXXJJXXJJXJJK (SEQ ID NO: 9, modified P14d min consensus),        wherein    -   X at position 2 can be M, L, E, Q, D, N, G, A, S, isoD, or        g-glutamate;    -   X at position 3 can be Q, N, E, D, G, A, S, isoD, or        g-glutamate;    -   X at position 6 can be Q, N, E, D, G, A, S, isoD, or        g-glutamate;    -   X at position 7 can be Q, N, E, D, G, A, S, isoD, or        g-glutamate;    -   X at position 10 can be K, Q, N, E, D, R, G, A, or S; and        J at positions 1, 4, 5, 8, 9, 11, and 12 are hydrophobic amino        acids selected from L, V, I, and F, except that at least one of        the amino acids at these positions is a non-hydrophobic amino        acid or A;        wherein the peptide induces an active plant response, but does        not induce a hypersensitive response, when applied to plant        tissue. Preferably, one to four, not more than three, not more        than two, or exactly one of the residues at positions 7, 10, 11,        14, 15, 17, and 18 of SEQ ID NO: 8 is a non-hydrophobic amino        acid or A; and one to four, not more than three, not more than        two, or exactly one of the residues at positions 1, 4, 5, 8, 9,        11, and 12 of SEQ ID NO: 9 is a non-hydrophobic amino acid or A.        In one embodiment, not more than one of the J residues is A, and        preferably the A residue is at one of positions 7, 10, 11, 14,        17, and 18 of SEQ ID NO: 8 or positions 1, 4, 5, 8, 11, and 12        of SEQ ID NO: 9; one or two of the remaining J residues is        optionally a non-hydrophobic amino acid, and all other J        residues are L, I, V, or F. In certain embodiments, not more        than one of the J residues is A, and preferably the A residue is        at one of positions 7, 10, 11, 14, 17, and 18 of SEQ ID NO: 8 or        positions 1, 4, 5, 8, 11, and 12 of SEQ ID NO: 9; and all other        J residues are L, I, V, or F.

Exemplary peptides that share the consensus structure with SEQ ID NO: 8or 9 are identified in Table 11 below:

TABLE 11 Peptide Variants of P14d (consensus SEQ ID NO: 8 or 9) NameSequence SEQ ID NO: P14d-7D  QDPMQADMQLLEDLVKLLK 208 P14d-10DQDPMQALMQDLEDLVKLLK 209 P14d-11D QDPMQALMQLDEDLVKLLK 210 P14d-14DQDPMQALMQLLEDDVKLLK 211 P14d-15D QDPMQALMQLLEDLDKLLK 212 P14d-17DQDPMQALMQLLEDLVKDLK 213 P14d-18D QDPMQALMQLLEDLVKLDK 214 P14d-7S QDPMQASMQLLEDLVKLLK 215 P14d-10S QDPMQALMQSLEDLVKLLK 216 P14d-11SQDPMQALMQLSEDLVKLLK 217 P14d-14S QDPMQALMQLLEDSVKLLK 218 P14d-15SQDPMQALMQLLEDLSKLLK 219 P14d-17S QDPMQALMQLLEDLVKSLK 220 P14d-18SQDPMQALMQLLEDLVKLSK 221

Additional variants of these peptides may include the substitution ofmethionine at one or both of positions 4 and 8 with L, E, Q, D, N, G, A,S, isoD, or g-glutamate, as noted above.

In certain other embodiments, the isolated peptide consists essentiallyof, or consists of, the amino acid sequence of one or more of SEQ IDNOS: 208-221.

In this embodiment, the isolated peptide is preferably stable whendissolved in water; resistant to chemical degradation in aqueousconditions in the presence of a pH buffer and a biocide, as describedabove; and/or has a solubility in an aqueous solution of at least about1.0 mg/ml.

A further aspect of the invention relates to a peptide having the aminoacid sequence of:

(i) JXXJJXJJXXJJ (SEQ ID NO: 10, modified P25 consensus) wherein

-   -   X at position 2 can be Q, N, E, g-glutamate, D, isoD, T, S, A,        or G;    -   X at position 3 can be K, Q, N, E, g-glutamate, D, isoD, T, S,        A, or G;    -   X at position 6 can be K, Q, N, E, g-glutamate, D, isoD, T, S,        A, or G;    -   X at position 9 can be E, g-glutamate, D, isoD, Q, N, T, S, A,        or G;    -   X at position 10 can be A, G, S, T, E, g-glutamate, D, isoD, Q,        or N; and    -   J at positions 1, 4, 5, 7, 8, 10, and 11 are hydrophobic amino        acids selected from L, V, I, F, and M, except that at least one        of the amino acids at these positions is a non-hydrophobic amino        acid or A; or        (ii) JXXJJXXJJXJJXXJJ (SEQ ID NO: 11, modified P25 consensus)        wherein    -   X at position 2 can be T, S, A, G, D, isoD, E, g-glutamate, Q,        or N;    -   X at position 3 can be G, T, S, A, D, isoD, E, g-glutamate, Q,        or N;    -   X at position 6 can be Q, N, E, g-glutamate, D, isoD, T, S, A,        or G;    -   X at position 7 can be K, Q, N, E, g-glutamate, D, isoD, T, S,        A, or G;    -   X at position 10 can be K, Q, N, E, g-glutamate, D, isoD, T, S,        A, or G;    -   X at position 13 can be E, g-glutamate, D, isoD, Q, N, T, S, A,        or G;    -   X at position 14 can be A, G, S, T, E, g-glutamate, D, isoD, Q,        or N;    -   J at positions 1, 4, 5, 8, 9, 11, 12, and 15 are hydrophobic        amino acids selected from L, V, I, F, and M, except that at        least one of the amino acids at these positions is a        non-hydrophobic amino acid or A; and    -   J at position 16 is optional and a hydrophobic amino acid        selected from L, V, I, and F;        wherein the peptide induces an active plant response, but does        not induce a hypersensitive response, when applied to plant        tissue. Preferably, one to four, not more than three, not more        than two, or exactly one of the residues at positions 1, 4, 5,        7, 8, 10, and 11 of SEQ ID NO: 10 is a non-hydrophobic amino        acid, and one to four, not more than three, not more than two,        or exactly one of the residues at positions 1, 4, 5, 8, 9, 11,        12, 15, and 16 of SEQ ID NO: 11 is a non-hydrophobic amino acid.        In one embodiment, not more than one of the J residues is A, and        preferably the A residue is at one of positions 1, 4, 5, 7, 10,        and 11 of SEQ ID NO: 10 or positions 1, 4, 5, 8, 11, 12, 15, and        16 of SEQ ID NO: 11; one or two of the remaining J residues is        optionally a non-hydrophobic amino acid, and all other J        residues are L, I, V, or F. In certain embodiments, not more        than one of the J residues is A, and preferably the A residue is        at one of positions 1, 4, 5, 7, 10, and 11 of SEQ ID NO: 10 or        positions 1, 4, 5, 8, 11, 12, 15, and 16 of SEQ ID NO: 11; and        all other J residues are L, I, V, or F.

Exemplary peptides that share the consensus structure with SEQ ID NO: 10or 11 are identified in Table 12 below:

TABLE 12 Peptide Variants of P25 (consensus SEQ ID NO: 10 or 11) NameSequence SEQ ID NO: P25-5D  GGLTDTGVLQKLMKILNALVQ 222 P25-8D GGLTLTGDLQKLMKILNALVQ 223 P25-9D  GGLTLTGVDQKLMKILNALVQ 224 P25-12DGGLTLTGVLQKDMKILNALVQ 225 P25-13D GGLTLTGVLQKLDKILNALVQ 226 P25-15DGGLTLTGVLQKLMKDLNALVQ 227 P25-16D GGLTLTGVLQKLMKIDNALVQ 228 P25-19DGGLTLTGVLQKLMKILNADVQ 229 P25-20D GGLTLTGVLQKLMKILNALDQ 230 P25-5S GGLTSTGVLQKLMKILNALVQ 231 P25-8S  GGLTLTGSLQKLMKILNALVQ 232 P25-9S GGLTLTGVSQKLMKILNALVQ 233 P25-12S GGLTLTGVLQKSMKILNALVQ 234 P25-13SGGLTLTGVLQKLSKILNALVQ 235 P25-15S GGLTLTGVLQKLMKSLNALVQ 236 P25-16SGGLTLTGVLQKLMKISNALVQ 237 P25-19S GGLTLTGVLQKLMKILNASVQ 238 P25-20SGGLTLTGVLQKLMKILNALSQ 239

Additional variants of these peptides may include the substitution ofmethionine at position 13 with L, V, I, and F, as noted above.

In certain other embodiments, the isolated peptide consists essentiallyof, or consists of, the amino acid sequence of one or more of SEQ IDNOS: 222-239.

In this embodiment, the isolated peptide is preferably stable whendissolved in water; resistant to chemical degradation in aqueousconditions in the presence of a pH buffer and a biocide, as describedabove; and/or has a solubility in an aqueous solution of at least about1.0 mg/ml.

Still another aspect of the invention relates to a peptide having theamino acid sequence:

(i) XXXXXXXXXXXJXXJJXXJJXXJJXXX (SEQ ID NO: 12, modified P17/18),wherein

-   -   X at position 1 can be any amino acid, but preferably Q, S, E,        g-glutamate, A, T, G, D, isoD, N, K, or R;    -   X at position 2 can be any amino acid, but preferably Q, S, E,        g-glutamate, A, T, G, D, isoD, N, K, or R;    -   X at position 3 can be any amino acid, but preferably P, Q, S,        E, g-glutamate, A, T, G, D, isoD, N, K, or R;    -   X at position 4 can be any amino acid, but preferably I, Q, S,        E, g-glutamate, A, T, G, D, N, isoD, K, or R;    -   X at position 5 can be any amino acid, but preferably D, isoD,        S, E, g-glutamate, A, T, G, N, Q, K, or R;    -   X at position 6 can be any amino acid, but preferably R, Q, S,        E, g-glutamate, A, T, G, D, isoD, N, or K;    -   X of position 7 can be any amino acid, but preferably Q, S, E,        g-glutamate, A, T, G, D, isoD, N, K, or R;    -   X at position 8 can be any amino acid, but preferably T, Q, S,        E, g-glutamate, A, G, D, isoD, N, K, or R;    -   X at position 9 can be any amino acid, but preferably I, Q, S,        E, g-glutamate, A, T, G, D, isoD, N, K, or R;    -   X at position 10 can be any amino acid, but preferably E,        g-glutamate, Q, S, A, T, G, D, isoD, N, K, or R;    -   X at position 11 can be any amino acid, but preferably Q, S, E,        g-glutamate, A, T, G, D, isoD, N, K, or R;    -   X at position 13 can be any amino acid, but preferably A, S, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 14 can be any amino acid, but preferably Q, A, S,        T, G, D, isoD, E, g-glutamate, N, K, or R;    -   X at position 17 can be any amino acid, but preferably A, S, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 18 can be any amino acid, but preferably Q, A, S,        T, G, D, isoD, E, g-glutamate, N, K, or R;    -   X at position 21 can be any amino acid, but preferably K, A, S,        T, G, D, isoD, E, g-glutamate, Q, N, or R;    -   X at position 22 can be any amino acid, but preferably S, A, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 25 can be any amino acid, but preferably S, A, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 26 can be any amino acid, but preferably P, S, A,        T, G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 27 can be any amino acid, but preferably Q, S, A,        T, G, D, isoD, E, g-glutamate, N, K, or R; and    -   J at positions 12, 15, 16, 19, 20, 23, and 24 are hydrophobic        amino acids selected from L, V, I, F, and M, except that at        least one of the amino acids at these positions is a        non-hydrophobic amino acid or A;    -   or        (ii) JXXJJXXJJXXJJ (SEQ ID NO: 13, modified P17/18 min        consensus), wherein    -   X at position 2 can be any amino acid, but preferably A, S, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 3 can be any amino acid, but preferably Q, A, S,        T, G, D, isoD, E, g-glutamate, N, K, or R;    -   X at position 6 can be any amino acid, but preferably A, S, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 7 can be any amino acid, but preferably Q, A, S,        T, G, D, isoD, E, g-glutamate, N, K, or R;    -   X at position 10 can be any amino acid, but preferably K, A, S,        T, G, D, isoD, E, g-glutamate, Q, N, or R;    -   X at position 11 can be any amino acid, but preferably S, A, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R; and    -   J at positions 1, 4, 5, 8, 9, 12, and 13 are hydrophobic amino        acids selected from L, V, I, F, and M, except that at least one        of the amino acids at these positions is a non-hydrophobic amino        acid or A;        wherein the peptide induces an active plant response, but does        not induce a hypersensitive response, when applied to plant        tissue. Preferably, one to four, not more than three, not more        than two, or exactly one of the residues at positions 12, 15,        16, 19, 20, 23, and 24 of SEQ ID NO: 12 is a non-hydrophobic        amino acid or A; and one to four, not more than three, not more        than two, or exactly one of the residues at positions 1, 4, 5,        8, 9, 12, and 13 of SEQ ID NO: 13 is a non-hydrophobic amino        acid or A. In one embodiment, not more than one of the J        residues is A, and preferably the A residue is at one of        positions 12, 15, 16, 19, 23, and 24 of SEQ ID NO: 12 or        positions 1, 4, 5, 8, 12, and 13 of SEQ ID NO: 13; one or two of        the remaining J residues is optionally a non-hydrophobic amino        acid, and all other J residues are L, I, V, F, or M. In certain        embodiments, not more than one of the J residues is A, and        preferably the A residue is at one of positions 12, 15, 16, 19,        23, and 24 of SEQ ID NO: 12 or positions 1, 4, 5, 8, 12, and 13        of SEQ ID NO: 13; and all other J residues are L, I, V, F, or M.

Exemplary peptides that share the consensus structure with SEQ ID NO: 12or 13, or are derived from SEQ ID NO: 12 or 13 and meet the consensusstructure of SEQ ID NO: 1, are identified in Table 13 below:

TABLE 13 Peptide Variants of P17/P18 (consensus SEQ ID NO: 12 or 13)Name Sequence SEQ ID NO: P18min-7D  SEEEEE DAQLLAQLLKSLL 85 P18min-10DSEEEEELAQDLAQLLKSLL 86 P18min-11D SEEEEELAQLDAQLLKSLL 87 P18min-14DSEEEEELAQLLAQDLKSLL 88 P18min-15D SEEEEELAQLLAQLDKSLL 89 P18min-18DSEEEEELAQLLAQLLKSDL 90 P18min-19D SEEEEELAQLLAQLLKSLD 91 P18min-7S SEEEEE SAQLLAQLLKSLL 92 P18min-10S SEEEEELAQSLAQLLKSLL 93 P18min-11SSEEEEELAQLSAQLLKSLL 94 P18min-14S SEEEEELAQLLAQSLKSLL 95 P18min-15SSEEEEELAQLLAQLSKSLL 96 P18min-18S SEEEEELAQLLAQLLKSSL 97 P18min-19SSEEEEELAQLLAQLLKSLS 98In Table 13, peptides include the solubility tag SEEEEE, indicated byitalic print. Peptides comprising the sequences shown in Table 13 butlacking this specific solubility tag (or having a different solubilitytag) are also contemplated herein.

In certain other embodiments, the isolated peptide consists essentiallyof, or consists of, the amino acid sequence of one or more of SEQ IDNOS: 85-98.

In this embodiment, the isolated peptide is preferably stable whendissolved in water; resistant to chemical degradation in aqueousconditions in the presence of a pH buffer and a biocide, as describedabove; and/or has a solubility in an aqueous solution of at least about1.0 mg/ml.

Yet another aspect of the invention relates to a peptide having theamino acid sequence:

XJXXJJXJJXXJJ (SEQ ID NO: 14, modified P19 consensus), wherein

-   -   X at position 1 is optional and can be L, I, V, F, or M;    -   X at position 3 can be any amino acid, but preferably K, A, S,        T, G, D, isoD, E, g-glutamate, Q, N, or R;    -   X at position 4 can be any amino acid, but preferably A, S, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 7 can be any amino acid, but preferably K, A, S,        T, G, D, isoD, E, g-glutamate, Q, N, or R;    -   X at position 10 can be any amino acid, but preferably A, S, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 11 can be any amino acid, but preferably R, A, S,        T, G, D, isoD, E, g-glutamate, Q, N, or K; and    -   J at positions 2, 5, 6, 8, 9, 12, and 13 are hydrophobic amino        acids selected from L, V, I, F, and M, except that at least one        of the amino acids at these positions is a non-hydrophobic amino        acid or A;        wherein the peptide induces an active plant response, but does        not induce a hypersensitive response, when applied to plant        tissue. Preferably, one to four, not more than three, not more        than two, or exactly one of the residues at positions 2, 5, 6,        8, 9, 12, and 13 is a non-hydrophobic amino acid. In one        embodiment, not more than one of the J residues is A, and        preferably the A residue is at one of positions 2, 5, 6, 8, 12,        and 13 of SEQ ID NO: 14; one or two of the remaining J residues        is optionally a non-hydrophobic amino acid, and all other J        residues are L, I, V, F, or M. In certain embodiments, not more        than one of the J residues is A, and preferably the A residue is        at one of positions 2, 5, 6, 8, 12, and 13 of SEQ ID NO: 14; and        all other J residues are L, I, V, F, or M.

Exemplary peptides that share the consensus structure with one of SEQ IDNO: 14, or are derived from one of SEQ ID NO: 14 and meet the consensusstructure of SEQ ID NO: 1, are identified in Table 14 below:

TABLE 14 Peptide Variants of P19 (consensus SEQ ID NO: 14) Name SequenceSEQ ID NO: P19min-7D  SEEEEE DKALLKLIARLL  99 P19min-10DSEEEEELKADLKLIARLL 100 P19min-11D SEEEEELKALDKLIARLL 101 P19min-13DSEEEEELKALLKDIARLL 102 P19min-14D SEEEEELKALLKLDARLL 103 P19min-17DSEEEEELKALLKLIARDL 104 P19min-18D SEEEEELKALLKLIARLD 105 Pl9min-7S SEEEEE SKALLKLIARLL 106 P19min-10S SEEEEELKASLKLIARLL 107 P19min-11SSEEEEELKALSKLIARLL 108 P19min-13S SEEEEELKALLKSIARLL 109 P19min-14SSEEEEELKALLKLSARLL 110 P19min-17S SEEEEELKALLKLIARSL 111 P19min-18SSEEEEELKALLKLIARLS 112In Table 14, peptides include the solubility tag SEEEEE, indicated byitalic print. Peptides comprising the sequences shown in Table 14 butlacking this specific solubility tag (or having a different solubilitytag) are also contemplated herein.

In certain embodiments, the isolated peptide consists essentially of, orconsists of, the amino acid sequence of SEQ ID NOS: 99-112.

In this embodiment, the isolated peptide is preferably stable whendissolved in water; resistant to chemical degradation in aqueousconditions in the presence of a pH buffer and a biocide, as describedabove; and/or has a solubility in an aqueous solution of at least about1.0 mg/ml.

A further aspect of the invention relates to an isolated peptide thatincludes the amino acid sequence of:

Z₁-LLXLFXXIL-Z₂ (SEQ ID NO: 126, P3 minimum) wherein

-   -   X at position 3 is any hydrophilic amino acid, preferably K, A,        S, T, G, D, isoD, E, g-glutamate, Q, N, or R;    -   X at position 6 is any hydrophilic amino acid, preferably K, A,        S, T, G, D, isoD, E, g-glutamate, Q, N, or R;    -   X at position 7 is any hydrophilic amino acid, preferably K, A,        S, T, G, D, isoD, E, g-glutamate, Q, N, or R; and    -   wherein one of Z₁ and Z₂ is present, but preferably not both,        with Z₁ comprising LXX- where each X is a hydrophilic amino        acid, preferably K, A, S, T, G, D, isoD, E, g-glutamate, Q, N,        or R, and with Z₂ comprising -XXLF where each X is a hydrophilic        amino acid, preferably K, A, S, T, G, D, isoD, E, g-glutamate,        Q, N, or R.        wherein the peptide induces an active plant response, but does        not induce a hypersensitive response, when applied to plant        tissue.

In one embodiment, Z₁ but not Z₂ is present. In an alternativeembodiment, Z₂ but not Z₁ is present.

Exemplary peptides that share the consensus structure with SEQ ID NO:126, or are derived from SEQ ID NO: 126 and meet the consensus structureof SEQ ID NO: 1, are identified in Table 15 below:

TABLE 15 Peptide Variants of P3 (consensus SEQ ID NO: 126) Name SequenceSEQ ID NO: P3-5   SEEELQQLLKLFSEIL 156 P3-8   SEEEEEELLKLFSEILQSLF 157P3-9   SEEEEQQLLKLFSEILQSLF 158 P3-10 SEEEEELQQLLKLFSEILQ 159In Table 15, peptides include the solubility tag SEEE, SEEEE, andSEEEEEE, indicated by italic print. Peptides comprising the sequencesshown in Table 15 but lacking this specific solubility tag (or having adifferent solubility tag) are also contemplated herein.

In certain embodiments, the isolated peptide consists essentially of, orconsists of, the amino acid sequence of SEQ ID NOS: 156-159.

In this embodiment, the isolated peptide is preferably stable whendissolved in water; resistant to chemical degradation in aqueousconditions in the presence of a pH buffer and a biocide, as describedabove; and/or has a solubility in an aqueous solution of at least about1.0 mg/ml.

Another aspect of the invention relates to an isolated peptide thatincludes the amino acid sequence of:

L-X-X-(L/I)-(L/I)-X-X-(L/I/V)-(L/I/V) (SEQ ID NO: 116)

wherein the peptide is free of cysteine and methionine; each X atpositions 2, 3, 6, 7 is any amino acid; and the peptide induces anactive plant response, but does not induce a hypersensitive response,when applied to plant tissue.

In certain embodiments, X at one or more of positions 2, 3, 6, and 7 isa non-hydrophobic amino acid, X at two or more of positions 2, 3, 6, and7 is a non-hydrophobic amino acid, X at three or more of positions 2, 3,6, and 7 is a non-hydrophobic amino acid, or X at each of positions 2,3, 6, and 7 is a non-hydrophobic amino acid. In this embodiment, thenon-hydrophobic amino acids independently may be any one of G, A, S, T,D, isoD, E, g-glutamate, Q, N, K, and R.

The peptides according to this aspect of the invention are generallydenoted by a truncation of the HR box sequence, which is described inco-pending U.S. patent application Ser. No. 14/872,298, entitled“Hypersensitive Response Elicitor Peptides and Use Thereof”, filed Oct.1, 2015, which is hereby incorporated by reference in its entirety.Thus, by virtue of the truncation of the HR box sequence, either bytermination of the peptide or substitution of amino acid residues thatform part of the HR box sequence, the isolated peptide according to thisaspect do not comprise the amino acid sequence:

(L/I/V/F)-X-X-(L/I/V/F)-(L/I)-X-X-(L/I/V)-(L/I)-X-X-(L/I/V/F)-(L/I/V/F)(SEQ ID NO: 125)

wherein each X at positions 2, 3, 6, 7, 10, 11 is any amino acid.

Exemplary peptides that share the consensus structure with SEQ ID NO:116 are identified in Table 16 below:

TABLE 16 SEQ Name Sequence ID NO: P14-22L,QAGPQSANKTGNVDDANNQDPLQALLQLLEDLV 115 26L P14-32                  SEEEEELEQLLEDLVKLL 124 P14-31                 SEEEEEALEQLLEDLVKLL 129 P14-39                  SEEEEELEQLLEDLV 160 P14-41                  SEEEEELLQLLEDLV 128 P1-29    NQGISEKQLDQLLTQLI 130P1-31       SEEEELDQLLTQLI 131 P4-111    SQGISEKQLDQLLSQLI 132 P4-115      SEEEELDQLLSQLI 139 P18-9      SEEEEE LAQLLAQLL 143 P19-12       SEEEEEKALLKLIARLL 146 P19-13         SEEEEEALLKLIARLL 147 P15-61   SEEEEEEEIAKLIS_LIESLL 151 P25-9      SEEEEELQKLLK_ILEALV 152In Table 16, peptides include the solubility tags SEEEE and SEEEEE,indicated by italic print. Peptides comprising the sequences shown inTable 15 but lacking the specific solubility tag (or having a differentsolubility tag) are also contemplated herein.

In certain embodiments, the isolated peptide consists essentially of, orconsists of, the amino acid sequence of SEQ ID NOS: 115, 124, 128-132,139, 143, 146, 147, 151, 152, and 160.

In this embodiment, the isolated peptide is preferably stable whendissolved in water; resistant to chemical degradation in aqueousconditions in the presence of a pH buffer and a biocide, as describedabove; and/or has a solubility in an aqueous solution of at least about1.0 mg/ml.

The isolated peptides of the invention can also be presented in the formof a fusion peptide that includes, in addition, a second amino acidsequence coupled to the inventive peptides via peptide bond. The secondamino acid sequence can be a purification tag, such as poly-histidine(His₆-), a glutathione-S-transferase (GST-), or maltose-binding protein(MBP-), which assists in the purification but can later be removed,i.e., cleaved from the peptide following recovery. Protease-specificcleavage sites or chemical-specific cleavage sites (i.e., in a cleavablelinker sequence) can be introduced between the purification tag and thedesired peptide. Protease-specific cleavage sites are well known in theliterature and include, without limitation, the enterokinase specificcleavage site (Asp)₄-Lys, which is cleaved after lysine; the factor Xaspecific cleavage site Ile-(Glu or Asp)-Gly-Arg, which is cleaved afterarginine; the trypsin specific cleavage site, which cleaves after Lysand Arg; and the Genenase™ I specific cleavage sitePro-Gly-Ala-Ala-His-Tyr. Chemicals and their specific cleavage sitesinclude, without limitation, cyanogen bromide (CNBr), which cleaves atmethionine (Met) residues; BNPS-skatole, which cleaves at tryptophan(Trp) residues; formic acid, which cleaves at aspartic acid-proline(Asp-Pro) peptide bonds; hydroxylamine, which cleaves atasparagine-glycine (Asn-Gly) peptide bonds; and2-nitro-5-thiocyanobenzoic acid (NTCB), which cleaves at cysteine (Cys)residues (see Crimmins et al., “Chemical Cleavage of Proteins inSolution,” Curr. Protocol. Protein Sci., Chapter 11:Unit 11.4 (2005),which is hereby incorporated by reference in its entirety). In order touse one of these cleavage methods, it may be necessary to removeunwanted cleavage sites from within the desired peptide sequences bymutation. For example, P14-22E,26E-R (SEQ ID NO: 23) has been mutatedfor compatibility with trypsin: the lysine residue at position 9 ismutated to a glutamate and a C-terminal arginine is added to representthe product of a theoretical trypsin cleavage. The desired peptideproduct can be purified further to remove the cleaved purification tags.

The isolated peptides of the invention can also be presented in the formof a fusion peptide that includes multiple peptide sequences of thepresent invention linked together by a linker sequence, which may or maynot take the form of a cleavable amino acid sequence of the typedescribed above. Such multimeric fusion proteins may or may not includepurification tags. In one embodiment, each monomeric sequence caninclude a purification tag linked to a peptide of the invention by afirst cleavable peptide sequence; and the several monomeric sequencescan be linked to adjacent monomeric sequences by a second cleavablepeptide sequence. Consequently, upon expression of the multimeric fusionprotein, i.e., in a host cell, the recovered fusion protein can betreated with a protease or chemical that is effective to cleave thesecond cleavable peptide sequence, thereby releasing individualmonomeric peptide sequences containing purification tags. Upon affinitypurification, the recovered monomeric peptide sequences can be treatedwith a protease or chemical that is effective to cleave the firstcleavable peptide sequence and thereby release the purification tag fromthe peptide of interest. The latter can be further purified using gelfiltration and/or HPLC as described infra.

These fusion proteins may include the sequences, identified above as SEQID NO: 15-18, which were previously identified in Haapalainen et al.,“Functional Mapping of Harpin HrpZ of Pseudomonas syringae Reveals theSites Responsible for Protein Oligomerization, Lipid Interactions, andPlant Defence Induction,” Mol. Plant Pathol. 12(2):151-66 (2011), whichis hereby incorporated by reference in its entirety.

According to one approach, the peptides of the present invention can besynthesized by standard peptide synthesis operations. These include bothFMOC (9-fluorenylmethyloxy-carbonyl) and tBoc (tert-butyloxy-carbonyl)synthesis protocols that can be carried out on automated solid phasepeptide synthesis instruments including, without limitation, the AppliedBiosystems 431 A, 433 A synthesizers and Peptide Technologies Symphonyor large scale Sonata or CEM Liberty automated solid phase peptidesynthesizers. The use of alternative peptide synthesis instruments isalso contemplated. Peptides prepared using solid phase synthesis arerecovered in a substantially pure form.

The peptides of the present invention may be also prepared by usingrecombinant expression systems followed by separation and purificationof the recombinantly prepared peptides. Generally, this involvesinserting an encoding nucleic acid molecule into an expression system towhich the molecule is heterologous (i.e., not normally present). One ormore desired nucleic acid molecules encoding a peptide of the inventionmay be inserted into the vector. The heterologous nucleic acid moleculeis inserted into the expression system or vector in proper sense (5′-3′)orientation and correct reading frame relative to the promoter and anyother 5′ and 3′ regulatory molecules. Representative nucleotidesequences for expression in bacteria and plant hosts and included inTable 17 below:

TABLE 17 Peptide & Optimized SEQ Host Nucleotide Sequence ID NO: P14CAAGCTGGACCTCAATCTGCTAATAA 240 A. thaliana GACTGGAAATGTTGATGATGCTAATAATCAAGATCCTATGCAAGCTCTTATG CAACTTCTTGAAGATCTTGTT P14CAGGCAGGTCCGCAGAGCGCAAATAA 241 E. coli AACCGGTAATGTTGATGATGCAAATAATCAGGATCCGATGCAGGCACTGATG CAGCTGCTGGAAGATCTGGTT P14d-10DCAAGATCCTATGCAAGCTCTTATGCA 242 A. thaliana AGATCTTGAAGATCTTGTTAAGCTTCTTAAG P14d-10D CAGGATCCGATGCAGGCACTGATGCA 243 E. coliGGATCTGGAAGATCTGGTTAAACTGC TGAAA

With knowledge of the encoded amino acid sequence listed herein and thedesired transgenic organism, additional codon-optimized DNA sequencesand RNA sequences can be generated with nothing more than routine skill.

Expression (including transcription and translation) of a peptide orfusion polypeptide of the invention by the DNA construct may beregulated with respect to the level of expression, the tissue type(s)where expression takes place and/or developmental stage of expression. Anumber of heterologous regulatory sequences (e.g., promoters andenhancers) are available for controlling the expression of the DNAconstruct. These include constitutive, inducible and regulatablepromoters, as well as promoters and enhancers that control expression ina tissue- or temporal-specific manner. Exemplary constitutive promotersinclude the raspberry E4 promoter (U.S. Pat. Nos. 5,783,393 and5,783,394, each of which is hereby incorporated by reference in itsentirety), the nopaline synthase (NOS) promoter (Ebert et al., Proc.Natl. Acad. Sci. (U.S.A.) 84:5745-5749 (1987), which is herebyincorporated by reference in its entirety), the octopine synthase (OCS)promoter (which is carried on tumor-inducing plasmids of Agrobacteriumtumefaciens), the caulimovirus promoters such as the cauliflower mosaicvirus (CaMV) 19S promoter (Lawton et al., Plant Mol. Biol. 9:315-324(1987), which is hereby incorporated by reference in its entirety) andthe CaMV 35S promoter (Odell et al., Nature 313:810-812 (1985), which ishereby incorporated by reference in its entirety), the figwort mosaicvirus 35S-promoter (U.S. Pat. No. 5,378,619, which is herebyincorporated by reference in its entirety), the light-inducible promoterfrom the small subunit of ribulose-1,5-bis-phosphate carboxylase(ssRUBISCO), the Adh promoter (Walker et al., Proc. Natl. Acad. Sci.(U.S.A.) 84:6624-6628 (1987), which is hereby incorporated by referencein its entirety), the sucrose synthase promoter (Yang et al., Proc.Natl. Acad. Sci. (U.S.A.) 87:4144-4148 (1990), which is herebyincorporated by reference in its entirety), the R gene complex promoter(Chandler et al., Plant Cell 1:1175-1183 (1989), which is herebyincorporated by reference in its entirety), the chlorophyll a/b bindingprotein gene promoter, the CsVMV promoter (Verdaguer et al., Plant MolBiol., 37:1055-1067 (1998), which is hereby incorporated by reference inits entirety), and the melon actin promoter (PCT Publ. No. WO00/56863,which is hereby incorporated by reference in its entirety). Exemplarytissue-specific promoters include the tomato E4 and E8 promoters (U.S.Pat. No. 5,859,330, which is hereby incorporated by reference in itsentirety) and the tomato 2AII gene promoter (Van Haaren et al., PlantMol Bio., 21:625-640 (1993), which is hereby incorporated by referencein its entirety).

In one preferred embodiment, expression of the DNA construct is undercontrol of regulatory sequences from genes whose expression isassociated with early seed and/or embryo development. Indeed, in apreferred embodiment, the promoter used is a seed-enhanced promoter.Examples of such promoters include the 5′ regulatory regions from suchgenes as napin (Kridl et al., Seed Sci. Res. 1:209:219 (1991), which ishereby incorporated by reference in its entirety), globulin (Belangerand Kriz, Genet. 129: 863-872 (1991), GenBank Accession No. L22295, eachof which is hereby incorporated by reference in its entirety), gammazein Z 27 (Lopes et al., Mol Gen Genet. 247:603-613 (1995), which ishereby incorporated by reference in its entirety), L3 oleosin promoter(U.S. Pat. No. 6,433,252, which is hereby incorporated by reference inits entirety), phaseolin (Bustos et al., Plant Cell 1(9):839-853 (1989),which is hereby incorporated by reference in its entirety), arcelin5(U.S. Application Publ. No. 2003/0046727, which is hereby incorporatedby reference in its entirety), a soybean 7S promoter, a 7Sa promoter(U.S. Application Publ. No. 2003/0093828, which is hereby incorporatedby reference in its entirety), the soybean 754 conglycinin promoter, a7Sα promoter (Beachy et al., EMBO J. 4:3047 (1985); Schuler et al.,Nucleic Acid Res. 10(24):8225-8244 (1982), each of which is herebyincorporated by reference in its entirety), soybean trypsin inhibitor(Riggs et al., Plant Cell 1(6):609-621 (1989), which is herebyincorporated by reference in its entirety), ACP (Baerson et al., PlantMol. Biol., 22(2):255-267 (1993), which is hereby incorporated byreference in its entirety), stearoyl-ACP desaturase (Slocombe et al.,Plant Physiol. 104(4):167-176 (1994), which is hereby incorporated byreference in its entirety), soybean a′ subunit of β-conglycinin (Chen etal., Proc. Natl. Acad. Sci. 83:8560-8564 (1986), which is herebyincorporated by reference in its entirety), Vicia faba USP (U.S.Application Publ. No. 2003/229918, which is hereby incorporated byreference in its entirety) and Zea mays L3 oleosin promoter (Hong etal., Plant Mol. Biol., 34(3):549-555 (1997), which is herebyincorporated by reference in its entirety).

Nucleic acid molecules encoding the peptides of the present inventioncan be prepared via solid-phase synthesis using, e.g., thephosphoramidite method and phosphoramidite building blocks derived fromprotected 2′-deoxynucleosides. To obtain the desired oligonucleotide,the building blocks are sequentially coupled to the growingoligonucleotide chain in the order required by the sequence of theproduct. Upon the completion of the chain assembly, the product isreleased from the solid phase to solution, deprotected, collected, andtypically purified using HPLC. The limits of solid phase synthesis aresuitable for preparing oligonucleotides up to about 200 nt in length,which encodes peptides on the order of about 65 amino acids or less. Theends of the synthetized oligonucleotide can be designed to includespecific restriction enzyme cleavage site to facilitate ligation of thesynthesized oligonucleotide into an expression vector.

For longer peptides, oligonucleotides can be prepared via solid phasesynthesis and then the synthetic oligonucleotide sequences ligatedtogether using various techniques. Recombinant techniques for thefabrication of whole synthetic genes are reviewed, for example, inHughes et al., “Chapter Twelve—Gene Synthesis: Methods andApplications,” Methods in Enzymology 498:277-309 (2011), which is herebyincorporated by reference in its entirety.

Once a suitable expression vector is selected, the desired nucleic acidsequences are cloned into the vector using standard cloning proceduresin the art, as described by Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y.(1989), or U.S. Pat. No. 4,237,224 to Cohen and Boyer, which are herebyincorporated by reference in their entirety. The vector is thenintroduced to a suitable host.

A variety of host-vector systems may be utilized to recombinantlyexpress the peptides of the present invention. Primarily, the vectorsystem must be compatible with the host used. Host-vector systemsinclude, without limitation, the following: bacteria transformed withbacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such asyeast containing yeast vectors; mammalian cell systems infected withvirus (e.g., vaccinia virus, adenovirus, etc.); insect cell systemsinfected with virus (e.g., baculovirus); and plant cells infected byAgrobacterium. The expression elements of these vectors vary in theirstrength and specificities. Depending upon the host-vector systemutilized, any one of a number of suitable transcription and translationelements can be used to carry out this and other aspects of the presentinvention.

Purified peptides may be obtained by several methods. The peptide ispreferably produced in purified form (preferably at least about 80% or85% pure, more preferably at least about 90% or 95% pure) byconventional techniques. Depending on whether the recombinant host cellis made to secrete the peptide into growth medium (see U.S. Pat. No.6,596,509 to Bauer et al., which is hereby incorporated by reference inits entirety), the peptide can be isolated and purified bycentrifugation (to separate cellular components from supernatantcontaining the secreted peptide) followed by sequential ammonium sulfateprecipitation of the supernatant. The fraction containing the peptide issubjected to gel filtration in an appropriately sized dextran orpolyacrylamide column to separate the peptides from other proteins. Ifnecessary, the peptide fraction may be further purified by HPLC.

Alternatively, if the peptide of interest of interest is not secreted,it can be isolated from the recombinant cells using standard isolationand purification schemes. This includes disrupting the cells (e.g., bysonication, freezing, French press, etc.) and then recovering thepeptide from the cellular debris. Purification can be achieved using thecentrifugation, precipitation, and purification procedures describedabove. The use of purification tags, described above, can simplify thisprocess.

In certain embodiments, purification is not required. Where purificationis not performed, cell-free lysates can be recovered followingcentrifugation for removal of cellular debris. The resulting cell-freelysate can be treated with heat for a sufficient amount of time todeactivate any native proteases in the recovered fraction, e.g., 10 minat 100° C. If desired, one or more of biocidal agents, proteaseinhibitors, and non-ionic surfactants can be introduced to such acell-free preparation (see U.S. Application Publ. No. 20100043095 toWei, which is hereby incorporated by reference in its entirety).

Once the peptides of the present invention are recovered, they can beused to prepare a composition that includes a carrier, and one or moreadditives selected from the group consisting of a bacteriocidal orbiocidal agent, a protease inhibitor, a non-ionic surfactant, afertilizer, an herbicide, an insecticide, a fungicide, a nematicide,biological inoculants, plant regulators, and mixtures thereof.

In certain embodiments, the compositions include greater than about 1 nMof the peptide, greater than about 10 nM of the peptide, greater thanabout 20 nM of the peptide, greater than about 30 nM of the peptide,greater than about 40 nM of the peptide, greater than about 50 nM of thepeptide, greater than about 60 nM of the peptide, greater than about 70nM of the peptide, greater than 80 about nM of the peptide, greater thanabout 90 nM of the peptide, greater than about 100 nM of the peptide,greater than about 150 nM of the peptide, greater than about 200 nM ofthe peptide, or greater than about 250 nM of the peptide. In certainembodiments, the compositions include less than about 1 nM of thepeptide. For example, certain peptides can be present at a concentrationof less than about 2 ng/ml, less than about 1.75 ng/ml, less than about1.5 ng/ml, less than about 1.25 ng/ml, less than about 1.0 ng/ml, lessthan about 0.75 ng/ml, less than about 0.5 ng/ml, less than about 0.25ng/ml, or even less than about 0.1 ng/ml.

Suitable carriers include water, aqueous solutions optionally containingone or more co-solvents, slurries, and solid carrier particles.Exemplary solid carriers include mineral earths such as silicates,silica gels, talc, kaolins, limestone, lime, chalk, bole, loess, clays,dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate,magnesium oxide, ground synthetic materials, and products of vegetableorigin, such as cereal meal, tree bark meal, wood meal and nutshellmeal, cellulose powders, starches and starch derivatives, as well asother mono-, di-, and poly-saccharides.

Suitable fertilizers include, without limitation, ammonium sulfate,ammonium phosphate, ammonium nitrate, ureas, and combinations thereof.

Suitable insecticides include, without limitation, members of theneonicotinoid class such as imidicloprid, clothianidin, andthiamethoxam; members of the organophosphate class such as chlorpyrifosand malathion; members of the pyrethroid class such as permethrin; othernatural insecticides such as nicotine, nornicotine, and pyrethrins;members of the carbamate class such as aldicarb, carbofuran, andcarbaryl; members of the macrocyclic lactone class such as variousabamectin, avermectin, and ivermectin products; members of the diamideclass such as chlorantraniliprole, cyantraniliprole, and flubendiamide;chitin synthesis inhibitors, particularly those of the benzoylurea classsuch as lufenuron and diflubenzuron; and any combination thereof,including combinations of two or more, three or more, or four or moreinsecticides. Additional insecticides are listed in the Compendium ofPesticide Common Names, which is database operated by Alan Wood andavailable in electronic form at the alanwood.net internet site.

Suitable fungicides include, without limitation, members of thestrobilurin class such as azoxystrobin, pyraclostrobin, trifloxystrobin,picoxystrobin, and fluoxastrobin; members of the triazole class such asipconazole, metconazole, tebuconazole, triticonazole, tetraconazole,difenoconazole, flutriafol, propiconazole and prothioconazole; membersof the succinate dehydrogenase class such as carboxin, fluxapyroxad,boscalid and sedaxane: members of the phenylamide class such asmetalaxyl, mefenoxam, benalaxyl, and oxadiyxl; members of thephenylpyrrole class such as fludioxonil; members of the phthalimideclass such as captan; members of the dithiocarbamate class such asmancozeb and thiram; members of the benzimidazole class such asthiabendazole; and any combination thereof, including combinations oftwo or more, three or more, or four or more fungicides. Additionalfungicides are listed in the Compendium of Pesticide Common Names, whichis a database operated by Alan Wood and available in electronic form atthe alanwood.net internet site.

Suitable nematicides include, without limitation, chemicals of thecarbamate class such as aldicarb, aldoxycarb, oxamyl, carbofuran, andcleothocarb; and chemicals of the organophosphate class such asthionazin, ethoprophos, fenamiphos, fensulfothion, terbufos, isazofos,and ebufos. Additional nematicides are listed in the Compendium ofPesticide Common Names, which is a database operated by Alan Wood andavailable in electronic form at the alanwood.net internet site.

Suitable bactericides include, without limitation, those based ondichlorophene and benzylalcohol hemi formal (Proxel® from ICI orActicide® RS from Thor Chemie and Kathon® MK from Rohm & Haas) andisothiazolinone derivatives such as alkylisothiazolinones andbenzisothiazolinones (Acticide® MBS from Thor Chemie; Proxel® GXL fromICI). Additional bactericides are listed in the Compendium of PesticideCommon Names, which is a database operated by Alan Wood and available inelectronic form at the alanwood.net internet site.

Suitable inoculants include, without limitation, Bradyrhizobium spp.,particularly Bradyrhizobium japonicum (BASF Vault® products), Bacillussubtilis, Bacillus firmus, Bacillus pumilis, Streptomyces lydicus,Trichoderma spp., Pasteuria spp., other cultures of rhizobial cells(BASF Nodulator® and Rhizo-Flo®), and any combination thereof, includingcombinations of two or more, three or more, or four or more inoculants.

Plant regulators are chemical substances, either natural or synthetic,that either stimulate or inhibit plant biochemical signaling. These areusually, but not exclusively, recognized by receptors on the surface ofthe cell, causing a cascade of reactions in the cell. Suitable plantregulators include, without limitation, ethephon; ethylene; salicylicacid; acetylsalicylic acid; jasmonic acid; methyl jasmonate; methyldihydrojasmonate; chitin; chitosan; abscisic acid; any auxin compound orinhibitor, including but not limited to (4-chlorophenoxy)acetic acid,(2,4-dichlorophenoxy)acetic acid, and 2,3,5-triiodobenzoic acid; anycytokinin, including but not limited to kinetin and zeatin;gibberellins; brassinolide; and any combination thereof, includingcombinations of two or more, three or more, or four or more regulators.

Other suitable additives include buffering agents, wetting agents,coating agents, and abrading agents. These materials can be used tofacilitate application of the compositions in accordance with thepresent invention. In addition, the compositions can be applied to plantseeds with other conventional seed formulation and treatment materials,including clays and polysaccharides.

Compositions or systems use for plant seed treatment include: one ormore of the peptides of the present invention, preferably though notexclusively one or more of peptides p4-14s-9a (SEQ ID NO: 57),p4-14s-12d (SEQ ID NO: 59), p14 (SEQ ID NO: 113), p14a (SEQ ID NO: 114),p14-22E,26E (SEQ ID NO: 23), p1-29 (SEQ ID NO: 130), and p4-14S-16S (SEQID NO: 55) in combination with one or more insecticides, nematicides,fungicides, other inoculants, or other plant regulators, includingcombinations of multiple insecticides, or multiple nematicides, multiplefungicides, multiple other inoculants, or multiple plant regulators.Suitable insecticides, nematicides, fungicides, inoculants, and plantregulators for these combination treatments include those identifiedabove. These compositions are presented in the form of a singlecomposition at the time of seed treatment. In contrast, a system usedfor seed treatment may involve multiple treatments, e.g., a compositioncontaining the peptides is used in one treatment and a compositioncontaining the one or more insecticides, nematicides, fungicides, plantregulators and/or bactericides, is used in a separate treatment. In thelatter embodiment, both of these treatments are carried out at about thesame time, i.e., before planting or at about the time of planting.

One such example includes one or more of peptides of the presentinvention, including (without limitation) one or more of peptidesp4-14s-9a (SEQ ID NO: 57), p4-14s-12d (SEQ ID NO: 59), p14 (SEQ ID NO:113), p14a (SEQ ID NO: 114), p14-22E,26E (SEQ ID NO: 23), p1-29 (SEQ IDNO: 130), and p4-14S-16S (SEQ ID NO: 55), in combination with one ofPoncho™ (clothianidin) available from Bayer Crop Science, Poncho™ VOTiVO(clothianidin and Bacillus firmus biological nematicide) available fromBayer Crop Science, and Gaucho™ (imidicloprid) available from Bayer CropScience.

Another example includes one or more of peptides of the presentinvention, including (without limitation) one or more of peptidesp4-14s-9a (SEQ ID NO: 57), p4-14s-12d (SEQ ID NO: 59), p14 (SEQ ID NO:113), p14a (SEQ ID NO: 114), p14-22E,26E (SEQ ID NO: 23), p1-29 (SEQ IDNO: 130), and p4-14S-16S (SEQ ID NO: 55), in combination with one ofCruiser™ (thiamethoxam) available from Syngenta, CruiserMaxx™(thiamethoxam, mefenoxam, and fludioxynil) available from Syngenta,Cruiser Extreme™ (thiamethoxam, mefenoxam, fludioxynil, andazoxystrobin) available from Syngenta, Avicta™ (thiamethoxam andabamectin) available from Syngenta, and Avicta™ Complete (thiamethoxam,abamectin, and Clariva Complete™ which contains the Pasteurianishizawae—Pn1 biological inoculant) available from Syngenta, and AvictaComplete™ Corn (thiamethoxam, mefenoxam, fludioxynil, azoxystrobin,thiabendazole and abamectin) available from Syngenta.

Another example includes one or more of peptides of the presentinvention, including (without limitation) one or more of peptidesp4-14s-9a (SEQ ID NO: 57), p4-14s-12d (SEQ ID NO: 59), p14 (SEQ ID NO:113), p14a (SEQ ID NO: 114), p14-22E,26E (SEQ ID NO: 23), p1-29 (SEQ IDNO: 130), and p4-14S-16S (SEQ ID NO: 55), in combination with one ofVault Liquid plus Integral (Bradyrhizobium species and Bacillus subtilisstrain MBI 600 inoculants) available from BASF, Vault NP (Bradyrhizobiumjaponicum inoculant) available from BASF, and Subtilex NG (Bacillussubtilis biological inoculant) available from BASF.

The present invention further relates to methods of imparting diseaseresistance to plants, enhancing plant growth, effecting pest control(including insects and nematodes), imparting biotic or abiotic stresstolerance to plants, and/or modulating plant biochemical signaling.These methods involve applying an effective amount of an isolatedpeptide of the invention, or a composition of the invention to a plantor plant seed or the locus where the plant is growing or is expected togrow. As a consequence of such application, the peptide contacts cellsof the plant or plant seed, and induces in the plant or a plant grownfrom the plant seed disease resistance, growth enhancement, tolerance tobiotic stress, tolerance to abiotic stress, or altered biochemicalsignaling. Alternatively, the peptide or composition of the inventioncan be applied to plants such that seeds recovered from such plantsthemselves are able to impart disease resistance in plants, to enhanceplant growth, to affect insect control, to impart tolerance to biotic orabiotic stress, and/or to modulate biochemical signaling.

In these embodiments, it is also possible to select plants or plantseeds or the locus to which the isolated peptide or composition of theinvention is applied. For example, for fields known to contain a highnematode content, the plants or plant seeds to be grown in such fields,or the fields (locus), can be selectively treated by applying theisolated peptide or composition of the invention as described herein;whereas no such treatment may be necessary for plants or plant seedsgrown in fields containing low nematode content. Similarly, for fieldshaving reduced irrigation, the plants or plant seeds to be grown in suchfields, or the fields (locus), can be selectively treated by applyingthe isolated peptide or composition of the invention as describedherein; whereas no such treatment may be necessary for plants or plantseeds grown in fields having adequate irrigation. Likewise, for fieldsprone to flooding, the plants or plant seeds to be grown in such fields,or the fields (locus), can be selectively treated by applying theisolated peptide or composition of the invention as described herein;whereas no such treatment may be necessary for plants or plant seedsgrown in fields that are not prone to flooding. As yet another exampleof such selection, for fields prone to insect attack at certain times ofthe growing season, the plants or plant seeds to be grown in suchfields, or the fields (locus), can be selectively treated by applyingthe isolated peptide or composition of the invention as describedherein; whereas the same field may not be treated at ineffective timesof the growing season or other fields that are not prone to such attackmay go untreated. Such selection steps can be carried out whenpracticing each of the methods of use described herein, i.e., impartingdisease resistance to plants, enhancing plant growth, effecting pestcontrol (including insects and nematodes), imparting biotic or abioticstress tolerance to plants, and/or modulating plant biochemicalsignaling.

As an alternative to applying an isolated peptide or a compositioncontaining the same to plants or plant seeds in order to impart diseaseresistance in plants, to effect plant growth, to control insects, toimpart stress resistance, and/or modulated biochemical signaling to theplants or plants grown from the seeds, transgenic plants or plant seedscan be utilized. When utilizing transgenic plants, this involvesproviding a transgenic plant transformed with a DNA molecule encoding apeptide of the invention and growing the plant under conditionseffective to permit that DNA molecule to impart disease resistance toplants, to enhance plant growth, to control insects, and/or to imparttolerance to biotic or abiotic stress. Alternatively, a transgenic plantseed transformed with a DNA molecule encoding a peptide of the inventioncan be provided and planted in soil. A plant is then propagated from theplanted seed under conditions effective to permit that DNA molecule toexpress the peptide and thereby impart disease resistance to thetransgenic plant, to enhance plant growth, to control insects, and/or toimpart tolerance to biotic or abiotic stress.

In these embodiments, it is also possible to select transgenic plants orplant seeds for carrying out the present invention. For example, forfields known to contain a high nematode content, the transgenic plantsor plant seeds can be selectively grown in such fields; whereasnon-transgenic plants or plant seeds can be grown in fields containinglow nematode content. Similarly, for fields having reduced irrigation,the transgenic plants or plant seeds can be selectively grown in suchfields; whereas non-transgenic plants or plant seeds can be grown infields having adequate irrigation. Likewise, for fields prone toflooding, the transgenic plants or plant seeds can be grown in suchfields; whereas non-transgenic plants or plant seeds can be grown infields that are not prone to flooding. As yet another example of suchselection, for fields prone to insect attack at certain times of thegrowing season, the transgenic plants or plant seeds can be selectivelygrown in such fields; whereas non-transgenic plants or plant seeds canbe grown in fields that are not prone to such insect attack. Suchselection steps can be carried out when practicing each of the methodsof use described herein, i.e., imparting disease resistance to plants,enhancing plant growth, effecting pest control (including insects andnematodes), imparting biotic or abiotic stress tolerance to plants,and/or modulating plant biochemical signaling.

The present invention further relates to methods of improvingdesiccation resistance for cuttings removed from ornamental plants,post-harvest disease resistance or desiccation resistance to fruit orvegetables harvested from plants, and/or improved longevity of fruit orvegetable ripeness for fruit or vegetables harvested from plants. Thesemethods involve applying an effective amount of an isolated peptide ofthe present invention or a composition according to the presentinvention to a plant or the locus where the plant is growing. As aconsequence of such application, the peptide contacts cells of the plantor plant seed, and induces desiccation resistance for cuttings removedfrom ornamental plants, post-harvest disease resistance or desiccationresistance to fruit or vegetables harvested from plants, and/or improvedlongevity of fruit or vegetable ripeness for fruit or vegetablesharvested from plants. Alternatively, an effective amount of an isolatedpeptide of the present invention or a composition according to thepresent invention can be applied to a harvested fruit or vegetable. As aconsequence of such application, the peptide contacts cells of theharvested fruit or vegetable, and induces post-harvest diseaseresistance or desiccation resistance to the treated fruit or vegetables,and/or improved longevity of fruit or vegetable ripeness for the treatedfruit or vegetables.

In these embodiments, it is also possible to select plants, cuttings,fruits, vegetables, or the locus to which the isolated peptide orcomposition of the invention is applied. For example, for harvestedcuttings or fruit or vegetables that are being shipped great distancesor stored for long periods of time, then these can be selectivelytreated by applying the isolated peptide or composition of the inventionas described herein; whereas harvested cuttings or fruit or vegetablesthat are being shipped locally and intended to be consumed withoutsubstantially periods of storage can be excluded from such treatment.

As an alternative to applying an isolated peptide or a compositioncontaining the same to plants or plant seeds in order to inducedesiccation resistance to cuttings removed from ornamental plants,post-harvest disease resistance or desiccation resistance to fruit orvegetables harvested from plants, and/or improved longevity of fruit orvegetable ripeness for fruit or vegetables harvested from plants,transgenic plants or plant seeds can be utilized. When utilizingtransgenic plants, this involves providing a transgenic planttransformed with a DNA molecule encoding a peptide of the invention andgrowing the plant under conditions effective to permit that DNA moleculeto induce desiccation resistance for cuttings removed from ornamentalplants, post-harvest disease resistance or desiccation resistance tofruit or vegetables harvested from the transgenic plants, and/orimproved longevity of fruit or vegetable ripeness for fruit orvegetables harvested from the transgenic plants. Alternatively, atransgenic plant seed transformed with a DNA molecule encoding a peptideof the invention can be provided and planted in soil. A plant is thenpropagated from the planted seed under conditions effective to permitthat DNA molecule to express the peptide and thereby induce desiccationresistance for cuttings removed from ornamental plants, post-harvestdisease resistance or desiccation resistance to fruit or vegetablesharvested from the transgenic plants, and/or improved longevity of fruitor vegetable ripeness for fruit or vegetables harvested from thetransgenic plants.

In these embodiments, it is also possible to select transgenic plants orplant seeds for carrying out the present invention. For example,transgenic plants or plant seeds can be selected for growing when it isknown that harvested cuttings or fruit or vegetables are intended to beshipped great distances or stored for long periods of time post-harvest;whereas non-transgenic plants or plant seeds can be selected for growingwhen it is known that harvested cuttings or fruit or vegetables areintended to be shipped locally and/or consumed without substantiallyperiods of storage.

Suitable plants include dicots and monocots, including agricultural,silvicultural, ornamental and horticultural plants, whether in a naturalor genetically modified form. Exemplary plants include, withoutlimitation, alfalfa, apple, apricot, asparagus, avocados, bananas,barley, beans, beech (Fagus spec.), begonia, birch, blackberry,blueberry, cabbage, camphor, canola, carrot, castor oil plant, cherry,cinnamon, citrus, cocoa bean, coffee, corn, cotton, cucumber, cucurbit,eucalyptus, fir, flax, fodder beet, fuchsia, garlic, geranium, grapes,ground nut, hemp, hop, juneberry, juncea (Brassica juncea), jute,lentil, lettuce, linseed, melon, mustard, nectarine, oak, oats, oilpalm, oil-seed rape, olive, onion, paprika, pea, peach, pear,pelargonium, peppers, petunia, pine (Pinus spec.), plum, poplar (Populusspec.), pome fruit, potato, rape, raspberry, rice, rubber tree, rye,sorghum, soybean, spinach, spruce, squash, strawberry, sugar beet, sugarcane, sunflower, tea, teak, tobacco, tomato, triticale, turf,watermelon, wheat and willow (Salix spec.), Arabidopsis thaliana,Saintpaulia, poinsettia, chrysanthemum, carnation, and zinnia.

With respect to modified biochemical signaling, this includes bothenhancement of certain plant biochemical pathways and diminishment ofcertain other plant biochemical pathways. Biochemical signaling pathwaysthat can be altered in accordance with the present invention includegene expression and protein production, production of metabolites, andproduction of signaling molecules/secondary metabolites. Exemplarybiochemical signaling pathways and their modifications include, withoutlimitation, induction of nitric oxide production, peroxide production,and other secondary metabolites; agonist of the ethylene signalingpathway and induction of ethylene-responsive gene expression (see Donget al., Plant Phys. 136:3628-3638 (2004); Li et al., Planta 239:831-46(2014); Chang et al., PLoS One 10, e0125498 (2015), each of which ishereby incorporated by reference in its entirety); agonist of thesalicylic acid signaling pathway and induction of salicylicacid-responsive gene expression (see Dong et al., Plant J. 20:207-215(1999), which is hereby incorporated by reference in its entirety);agonist of the abscisic acid pathway and induction of abscisicacid-responsive gene expression (see Dong et al., Planta 221: 313-327(2005), which is hereby incorporated by reference in its entirety);agonist of the gibberellin signaling pathway and induction ofgibberellin-responsive gene expression (see Li et al., Planta 239:831-46(2014), which is hereby incorporated by reference in its entirety);antagonist of jasmonic acid signaling and inhibiting expression ofjasmonic acid-responsive genes (see Dong et al., Plant Phys.136:3628-3638 (2004), which is hereby incorporated by reference in itsentirety); inducing protease inhibitor expression (see Laluk andMengiste, Plant J. 68:480-494 (2011); Xia et al., Chin. Sci. Bull 56:2351-2358 (2011), each of which is hereby incorporated by reference inits entirety); inducing reactive oxygen species production in planttissues; inducing immune-related and antimicrobial peptide production,such as, without limitation, peroxidase, superoxide dismutase,chitinase, and β-1,3-glucanase (Wang et al., J. Agric. Food Chem.59:12527-12533 (2011), which is hereby incorporated by reference in itsentirety); and inducing expansin gene expression and production (see Liet al., Planta 239:831-46 (2014), which is hereby incorporated byreference in its entirety).

With respect to disease resistance, absolute immunity against infectionmay not be conferred, but the severity of the disease is reduced andsymptom development is delayed. Lesion number, lesion size, and extentof sporulation of fungal pathogens are all decreased. This method ofimparting disease resistance has the potential for treating previouslyuntreatable diseases, treating diseases systemically which might not betreated separately due to cost, and avoiding the use of infectiousagents or environmentally harmful materials.

The method of imparting pathogen resistance to plants in accordance withthe present invention is useful in imparting resistance to a widevariety of pathogens including viruses, bacteria, and fungi. Resistance,inter alia, to the following viruses can be achieved by the method ofthe present invention: Tobacco mosaic virus and Tomato mosaic virus.Resistance, inter alia, to the following bacteria can also be impartedto plants in accordance with present invention: pathogenic Pseudomonasspp., pathogenic Erwinia spp., pathogenic Xanthomonas spp., andpathogenic Ralstonia spp. Plants can be made resistant, inter alia, tothe following fungi by use of the method of the present invention:Fusarium spp. and Phytophthora spp.

With regard to the use of the peptides or compositions of the presentinvention to enhance plant growth, various forms of plant growthenhancement or promotion can be achieved. This can occur as early aswhen plant growth begins from seeds or later in the life of a plant. Forexample, plant growth according to the present invention encompassesgreater yield, increased plant vigor, increased vigor of seedlings(i.e., post-germination), increased plant weight, increased biomass,increased number of flowers per plant, higher grain and/or fruit yield,increased quantity of seeds produced, increased percentage of seedsgerminated, increased speed of germination, increased plant size,decreased plant height (for wheat), greater biomass, more and biggerfruit, earlier fruit coloration, earlier bud, fruit and plantmaturation, more tillers or side shoots, larger leaves, delayed leafsenescence, increased shoot growth, increased root growth, alteredroot/shoot allocation, increased protein content, increased oil content,increased carbohydrate content, increased pigment content, increasedchlorophyll content, increased total photosynthesis, increasedphotosynthesis efficiency, reduced respiration (lower O₂ usage),compensation for yield-reducing treatments, increased durability ofstems (and resistance to stem lodging), increased durability of roots(and resistance to root lodging), better plant growth in low lightconditions, and combinations thereof. As a result, the present inventionprovides significant economic benefit to growers. For example, earlygermination and early maturation permit crops to be grown in areas whereshort growing seasons would otherwise preclude their growth in thatlocale. Increased percentage of seed germination results in improvedcrop stands and more efficient seed use. Greater yield, increased size,and enhanced biomass production allow greater revenue generation from agiven plot of land.

With regard to the use of the peptides or compositions of the presentinvention to control pests (including but not limited to insects andnematodes, which are biotic stressors), such pest control encompassespreventing pests from contacting plants to which the peptide orcomposition of the invention has been applied, preventing direct damageto plants by feeding injury, causing pests to depart from such plants,killing pests proximate to such plants, interfering with insect larvalfeeding on such plants, preventing pests from colonizing host plants,preventing colonizing insects from releasing phytotoxins, interferingwith egg deposition on host plants, etc. The present invention alsoprevents subsequent disease damage to plants resulting from pestinfection.

The present invention is effective against a wide variety of insects(biotic stressors). European corn borer is a major pest of corn (dentand sweet corn) but also feeds on over 200 plant species includinggreen, wax, and lima beans and edible soybeans, peppers, potato, andtomato plus many weed species. Additional insect larval feeding pestswhich damage a wide variety of vegetable crops include the following:beet armyworm, cabbage looper, corn ear worm, fall armyworm, diamondbackmoth, cabbage root maggot, onion maggot, seed corn maggot, pickleworm(melonworm), pepper maggot, and tomato pinworm. Collectively, this groupof insect pests represents the most economically important group ofpests for vegetable production worldwide. The present invention is alsoeffective against nematodes, another class of economically importantbiotic stressors. Soybean Cyst Nematode (Heterodera glycines) is a majorpest of soybeans. Reniform Nematode (Rotylenchulus reniformis) is amajor pest of cotton as can parasitize additional crop species, notablysoy and corn. Additional nematode pests include the root knot nematodesof the genus Meloidogyne (particularly in cotton, wheat, and barley),cereal cyst nematodes of the genus Heterodera (particularly in soy,wheat, and barley), root lesion nematodes of the genus Pratylenchus,seed gall nematodes of the genus Anguina (particularly in wheat, barley,and rye), and stem nematodes of the genus Ditylenchus. Other bioticstressors include arachnids, weeds, and combinations thereof.

With regard to the use of the peptides or compositions of the presentinvention to impart abiotic stress resistance to plants, such abioticstress encompasses any environmental factor having an adverse effect onplant physiology and development. Examples of such environmental stressinclude climate-related stress (e.g., drought, flood, frost, coldtemperature, high temperature, excessive light, and insufficient light),air pollution stress (e.g., carbon dioxide, carbon monoxide, sulfurdioxide, NO_(x), hydrocarbons, ozone, ultraviolet radiation, acidicrain), chemical (e.g., insecticides, fungicides, herbicides, heavymetals), nutritional stress (e.g., over- or under-abundance offertilizer, micronutrients, macronutrients, particularly potassium,nitrogen derivatives, and phosphorus derivatives), and improved healingresponse to wounding. Use of peptides of the present invention impartsresistance to plants against such forms of environmental stress.

A further aspect of the present invention relates to the use of thepeptides of the present invention as a safener in combination with oneor more of the active agents (i.e., in a composition or in separatecompositions) for the control of aquatic weeds in a body of water asdescribed in U.S. Publ. No. 20150218099 to Mann, which is herebyincorporated by reference in its entirety.

Yet another aspect of the present invention relates to the use of thepeptides of the present invention as a plant strengthener in acomposition for application to plants grown under conditions of reducedwater irrigation, which composition also includes at least oneantioxidant and at least one radiation manager, and optionally at leastone plant growth regulator, as described in U.S. Publ. No. 20130116119to Rees et al., which is hereby incorporated by reference in itsentirety.

The methods of the present invention involving application of thepeptide or composition can be carried out through a variety ofprocedures when all or part of the plant is treated, including leaves,stems, roots, propagules (e.g., cuttings), fruit, etc. This may (butneed not) involve infiltration of the peptide into the plant. Suitableapplication methods include high or low pressure spraying, injection,and leaf abrasion proximate to when peptide application takes place.When treating plant seeds, in accordance with the application embodimentof the present invention, the hypersensitive response elicitor proteinor polypeptide can be applied by low or high pressure spraying, coating,immersion (e.g., soaking), or injection. Other suitable applicationprocedures can be envisioned by those skilled in the art provided theyare able to effect contact of the hypersensitive response elicitorpolypeptide or protein with cells of the plant or plant seed. Oncetreated with the peptides or compositions of the present invention, theseeds can be planted in natural or artificial soil and cultivated usingconventional procedures to produce plants. After plants have beenpropagated from seeds treated in accordance with the present invention,the plants may be treated with one or more applications of the peptidesor compositions of the invention to impart disease resistance to plants,to enhance plant growth, to control insects on the plants, to impartbiotic or abiotic stress tolerance, to improve desiccation resistance ofremoved cuttings, to impart post-harvest disease resistance ordesiccation resistance to harvested fruit or vegetables, and/or improvedlongevity of fruit or vegetable ripeness for harvested fruit orvegetables.

The peptides or compositions of the invention can be applied to plantsor plant seeds in accordance with the present invention alone or in amixture with other materials. Alternatively, the peptides orcompositions can be applied separately to plants with other materialsbeing applied at different times.

In the alternative embodiment of the present invention involving the useof transgenic plants and transgenic seeds, a peptide of the inventionneed not be applied topically to the plants or seeds. Instead,transgenic plants transformed with a DNA molecule encoding a peptide ofthe invention are produced according to procedures well known in theart. A vector suitable for expression in plants (i.e., containingtranslation and transcription control sequences operable in plants) canbe microinjected directly into plant cells by use of micropipettes totransfer mechanically the recombinant DNA. Crossway, Mol. Gen. Genetics,202:179-85 (1985), which is hereby incorporated by reference in itsentirety. The genetic material may also be transferred into the plantcell using polyethylene glycol. Krens, et al., Nature, 296:72-74 (1982),which is hereby incorporated by reference in its entirety.

Another approach to transforming plant cells with a gene encoding thepeptide of the invention is particle bombardment (also known asbiolistic transformation) of the host cell. This can be accomplished inone of several ways. The first involves propelling inert or biologicallyactive particles at cells. This technique is disclosed in U.S. Pat. Nos.4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., which arehereby incorporated by reference. Generally, this procedure involvespropelling inert or biologically active particles at the cells underconditions effective to penetrate the outer surface of the cell and tobe incorporated within the interior thereof. When inert particles areutilized, the vector can be introduced into the cell by coating theparticles with the vector containing the heterologous DNA.Alternatively, the target cell can be surrounded by the vector so thatthe vector is carried into the cell by the wake of the particle.Biologically active particles (e.g., dried bacterial cells containingthe vector and heterologous DNA) can also be propelled into plant cells.

Yet another method of introduction is fusion of protoplasts with otherentities, either minicells, cells, lysosomes or other fusiblelipid-surfaced bodies. Fraley, et al., Proc. Natl. Acad. Sci. USA,79:1859-63 (1982), which is hereby incorporated by reference in itsentirety. The DNA molecule may also be introduced into the plant cellsby electroporation. Fromm et al., Proc. Natl. Acad. Sci. USA, 82:5824(1985), which is hereby incorporated by reference in its entirety. Inthis technique, plant protoplasts are electroporated in the presence ofplasmids containing the expression cassette. Electrical impulses of highfield strength reversibly permeabilize biomembranes allowing theintroduction of the plasmids. Electroporated plant protoplasts reformthe cell wall, divide, and regenerate.

Another method of introducing the DNA molecule into plant cells is toinfect a plant cell with Agrobacterium tumefaciens or A. rhizogenespreviously transformed with the gene. Under appropriate conditions knownin the art, the transformed plant cells are grown to form shoots orroots, and develop further into plants. Generally, this procedureinvolves inoculating the plant tissue with a suspension of bacteria andincubating the tissue for 48 to 72 hours on regeneration medium withoutantibiotics at 25-28° C. Agrobacterium is a representative genus of thegram-negative family Rhizobiaceae. Its species are responsible for crowngall (A. tumefaciens) and hairy root disease (A. rhizogenes). The plantcells in crown gall tumors and hairy roots are induced to produce aminoacid derivatives known as opines, which are catabolized only by thebacteria. The bacterial genes responsible for expression of opines are aconvenient source of control elements for chimeric expression cassettes.In addition, assaying for the presence of opines can be used to identifytransformed tissue. Heterologous genetic sequences can be introducedinto appropriate plant cells, by means of the Ti plasmid of A.tumefaciens or the Ri plasmid of A. rhizogenes. The Ti or Ri plasmid istransmitted to plant cells on infection by Agrobacterium and is stablyintegrated into the plant genome. J. Schell, Science, 237:1176-83(1987), which is hereby incorporated by reference in its entirety.

After transformation, the transformed plant cells must be regenerated.Plant regeneration from cultured protoplasts is described in Evans etal., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co.,New York, 1983); and Nasil I. R. (ed.), Cell Culture and Somatic CellGenetics of Plants, Acad. Press, Orlando, Vol. 1, 1984, and Vol. III(1986), which are hereby incorporated by reference in their entirety.

It is known that practically all plants can be regenerated from culturedcells or tissues. Means for regeneration vary from species to species ofplants, but generally a suspension of transformed protoplasts or a petriplate containing transformed explants is first provided. Callus tissueis formed and shoots may be induced from callus and subsequently rooted.Alternatively, embryo formation can be induced in the callus tissue.These embryos germinate as natural embryos to form plants. The culturemedia will generally contain various amino acids and hormones, such asauxin and cytokinins. It is also advantageous to add glutamic acid andproline to the medium, especially for such species as corn and alfalfa.Efficient regeneration will depend on the medium, on the genotype, andon the history of the culture. If these three variables are controlled,then regeneration is usually reproducible and repeatable.

After the expression cassette is stably incorporated in transgenicplants, it can be transferred to other plants by sexual crossing. Any ofa number of standard breeding techniques can be used, depending upon thespecies to be crossed.

Once transgenic plants of this type are produced, the plants themselvescan be cultivated in accordance with conventional procedure with thepresence of the gene encoding the hypersensitive response elicitorresulting in disease resistance, enhanced plant growth, control ofinsects on the plant, abiotic or biotic stress tolerance, improveddesiccation resistance of removed cuttings, post-harvest diseaseresistance or desiccation resistance in harvested fruit or vegetables,and/or improved longevity of fruit or vegetable ripeness for harvestedfruit or vegetables.

Alternatively, transgenic seeds are recovered from the transgenicplants. These seeds can then be planted in the soil and cultivated usingconventional procedures to produce transgenic plants. The transgenicplants are propagated from the planted transgenic seeds under conditionseffective to impart disease resistance to plants, to enhance plantgrowth, to control insects, to impart abiotic or biotic stresstolerance, to improve desiccation resistance of removed cuttings, toimpart post-harvest disease resistance or desiccation resistance inharvested fruit or vegetables, and/or to impart improved longevity offruit or vegetable ripeness for harvested fruit or vegetables.

When transgenic plants and plant seeds are used in accordance with thepresent invention, they additionally can be treated with the samematerials as are used to treat the plants and seeds to which a peptideof the invention or composition of the invention is applied. These othermaterials, including peptides or composition of the invention, can beapplied to the transgenic plants and plant seeds by the above-notedprocedures, including high or low pressure spraying, injection, coating,and immersion. Similarly, after plants have been propagated from thetransgenic plant seeds, the plants may be treated with one or moreapplications of the peptides or compositions of the invention to impartdisease resistance, enhance growth, control insects, abiotic or bioticstress tolerance, desiccation resistance of removed cuttings,post-harvest disease resistance or desiccation resistance in harvestedfruit or vegetables, and/or improved longevity of fruit or vegetableripeness for harvested fruit or vegetables.

Such transgenic plants may also be treated with conventional planttreatment agents, e.g., bacteriocidal or biocidal agents, proteaseinhibitors, non-ionic surfactants, fertilizers, herbicides,insecticides, fungicides, nematicides, biological inoculants, plantregulators, and mixtures thereof, as described above.

EXAMPLES

The following examples are provided to illustrate embodiments of thepresent invention but are by no means intended to limit its scope.

Example 1 Hypersensitive Response Analysis

HR in tobacco was tested as described in Wei, Science 257:85-88 (1992),which is hereby incorporated by reference in its entirety. Briefly,peptides were dissolved at a concentration of 500 μg/ml in aqueoussolution. Four serial dilutions were performed with an equal volume ofwater, yielding peptide samples at 500, 250, 125, 62.5, 31.25 μg/mlpeptide solutions. Nicotiana tabacum cultivar xanthi plants were used at5-7 weeks old (preflowering). Leaves were lightly punctured with atoothpick in a middle leaf panel. Peptide solutions were then infusedvia needle-less syringe into the wound, filling the panel. Each peptidesample was infused into a leaf of 2 different plants. The leaves wereobserved and scored over the next 48 hours for withering and browning,lesions typical of programmed cell death.

Hypersensitive Response testing was performed on many peptides anddetermined to be negative. Some peptides have yet to be tested, designedas “to be determined” or “TBD” in Table 18 below, but are expected to beHR-negative based on the results for closely related peptides.

TABLE 18 Summary of Hypersensitive Response Results SEQ SEQ ID TobaccoID Tobacco Name NO: HR Name NO: HR P1-13P-20P 164 — P14 113 — P1-29 130TBD P14-22L,26L 115 — P1-31 131 TBD P14-22L,26E 19 — P3-5 156 —P14-22A,26L 20 TBD P3-9 158 — P14-22A,26E 21 TBD P3-10 159 — P14-22E,26L22 TBD P4-14S-9A 57 — P14-22E,26E 23 TBD P4-14S-9D 58 — P14-22L,26A 26 —P4-14S-9S 52 — P14-22A,26A 27 TBD P4-14S-9Y 53 — P14-22L,25-26A 28 TBDP4-14S-12D 59 — P14-22L,28-29A 29 TBD P4-14s-13D 48 — P14-22L,32-33A 30TBD P4-14S-13F 134 — P14-22L,26,29A 31 TBD P4-14S-13V 133 —P14-22L,28,32A 32 TBD P4-14S-13Q 54 — P14-dc2 122 TBD P4-14S-16S 55 —P14-dc4 123 TBD P4-14S-17D 49 — P14a 114 — P4-14S-20S 56 — P15-60 150 —P4-14S-21D 50 — P15-61 151 — P4-d10,14,18 62 — P15-62 148 TBD P4-d18 51— P15-63 149 TBD P4- 68 — P18-8 142 TBD i10A,14A,18A P4-i10A 65 — P18-9143 TBD P4-i14A 66 — P19-9 144 — P4-i18A 67 — P19-10 145 — P4-i21Q 63 —P19-12 146 — P4-i21H 64 — P19-13 147 — P4-111 132 — P6a-15D 199 TBDP4-116 140 TBD P6a-11S 204 TBD P4-117 141 TBD P15b-12S 188 TBD P14d-7D208 TBD P14d-17D 213 TBD P14d-10D 209 TBD P14d-18D 214 TBD P14d-11D 210TBD P14d-7S 215 TBD P14d-14D 211 TBD P14f-17S 45 TBD P14d-15D 212 TBDTBD = To be determined.

Example 2 Peroxide Response Analysis

The ferric-xylenol orange complex assay for hydrogen peroxide (Gay etal., Analytical Biochemistry 273:149-55 (1999), which is herebyincorporated by reference in its entirety) was used to determineperoxide production by plant leaf tissue. Briefly, reagent A wasprepared by mixing 10 mg of ferrous ammonium sulfate with 133 uL ofsulfuric acid in a total volume of 1 mL with water. Reagent B wasprepared by mixing 2 g of sorbitol with 9.34 mg of xylenol orange in 80ml of water. 1M hydrochloric acid was slowly added until the solutionturned yellow. The solution was then diluted with water to a totalvolume of 100 mL, and stored in a dark bottle at 4° C.

Leaves from either tobacco plants (plants grown to 6 true leaves) or soyplants (plants grown to 6 true leaves) were used for the assay. The leafwas first removed from the plant and placed on a paper towel. 0.5 cmleaf tissue circles were then removed from the leaf using a leatherpunch tool. These discs were then placed into the wells of a 96-wellplate. 250 ml of water was added to each disc. The plate was coveredwith parafilm and rested for 12 hours in the dark. The excess water wasthen removed and the wells washed with an additional 250 uL of water.The washes were then removed from the wells and replaced with 105 uL ofa 0.5 ug/ml solution of peptide in water or water blank, generally inquadruplicate. Where peptide solubility was a problem, pH buffer wasadded to both the blank and peptide samples. Leaf discs were incubatedwith the peptides for one hour under fluorescent lighting. 50 ul of thesolution from each leaf sample was removed to a new well for chemicalanalysis.

Just prior to analysis, reagents A and B were freshly mixed in a 1:100ratio to produce the assay reagent. 200 ul of this mixture was added toeach well containing 50 ul of leaf-treated solution. The plate wasincubated in the dark for 20 minutes and the absorbance at 595 nm wasdetected. Results were analyzed using a two-tailed Student's T-test. Theresults were divided into several response classes. XR++ indicated apositive response with absorbance >0.05 units above the negative controland a Student's T-test p<0.05. XR+ indicated an absorbance <0.05 unitsabove the blank and a Student's T-test p<0.05. XR weak indicated aresponse >0.02 units above the negative control with a p-value that wasnot significant. XR− indicated an absorbance within 0.02 units of thenegative control. It should be noted that there can be significantbiological variability in the peroxide response due to plant age,location of the disc within the leaf, and other factors. As a result,p-values from the T-test are a guide and not an absolute inclusioncriterion.

The results of these studies are summarized in Table 19 below:

TABLE 19 Summary of Peroxide Response Name SEQ ID NO: Soy TobaccoP1-13P-20P 164 ++ − P1-29 130 ND + P1-31 131 + − P3-5 156 ND Weak+ P3-9158 ND ++ P3-10 159 ND ++ P4-14S-9A 57 ++ + P4-14S-9D 58 Weak+ +P4-14S-9S 52 + Weak+ P4-14S-9Y 53 + + P4-14S-12D 59 + + P4-14S-13D 48 NDWeak+ P4-14S-13F 134 + Weak+ P4-14S-13V 133 + + P4-14S-13Q 54 + Weak+P4-14S-16S 55 − ++ P4-14S-17D 49 ++ ++ P4-14S-20S 56 − ++ P4-14S-21D 50++ ++ P4-d10,14,18 62 ++ − P4-d18 51 + ++ P4-i10A,14A,18A 68 + − P4-i10A65 ++ ++ P4-i14A 66 ++ ++ P4-i18A 67 ++ ++ P4-116 140 + ++ P4-117 141++ + P14 113 + Weak+ P14-22L,26L 115 Weak+ − P14-22L,26E 19 ++ Weak+P14-22A,26L 20 + + P14-22A,26E 21 Weak+ − P14-22E,26L 22 ++ ++P14-22E,26E 23 + Weak+ P14-22L,26A 26 + + P14-22A,26A 27 ++ −P14-22L,25-26A 28 ++ − P14-22L,28-29A 29 Weak+ − P14-22L,32-33A 30 ++ +P14-22L,26,29A 31 ++ − P14-22L,28,32A 32 ++ − P14-dc2 122 + ++ P14-dc4123 ++ ++ P14a 114 ++ ++ P15-60 150 ++ ++ P15-61 151 ++ + P15-62 148 − −P15-63 149 − − P18-8 142 + + P18-9 143 + + P14d-7D 208 ND + P14d-10D 209ND ++ P14d-11D 210 ND + P14d-14D 211 ND ++ P14d-15D 212 ND ++ P14d-17D213 ND ++ P14d-18D 214 ND ++ P14d-7S 215 ND ++ P14f-17S 45 ND ++ P6a-15D199 ND + P6a-11S 204 ND − P15b-12S 188 ND ++ P15b-21S 193 ND ++P18min-11S 94 ND + P18min-7D 85 ND ++ P19min-10S 107 ND ++ P19min-11S108 ND + P19min-13S 109 ND ++ P25min-15S 81 ND ++ P25-9D 224 ND + P25-12153 + + P25-13 154 ND + P25-14 155 ND ++ ND: Not determined

In general, the vast majority of peptides tested XR+ in Soy. However,tobacco was more discriminating. Most peptides that were a singlemutation from an HR+ sequence were XR+ in tobacco. Even a secondmutation often still resulted in XR+ phenotype. However, tests on p14variants showed the mutation of three leucine residues to alaninegenerally resulted in a loss of XR elicitation. Likewise, C-terminaltruncated variants of p15 (p15-62 and p15-63) were XR− in tobacco.

Example 3 Root & Shoot Growth Analysis

Peptides were tested for biological effects on the allocation of growthresources to the shoot (above ground) and root (below ground). Peptideswere dissolved at 0.2, 2, or 5 μg/ml in a total volume of 100 mldeionized water. Corn or soybean seeds were then soaked for one hour inthe peptide solution. Untreated control (UTC) plants were soaked indeionized water. Clear plastic 300 ml beverage cups (Solo®, DartContainer Corporation) were prepared for planting by marking the bottomwith a cross, dividing the bottom into four equal quadrants. The cupswere then filled with Sunshine Mix #1 soil (SunGro Horticulture) sievedto ¼″. 100 ml of water was added to the soil. Treated seeds were thenplanted by pressing the seed lightly into the top of the soil. The seedswere then covered with an additional 50 ml of loose soil. Seeds wereallowed to germinate and grow for 12-14 days.

The length of the shoot was measured as the distance from the soil tothe lightly stretched tip of the highest leaf for each plant. Plantsthat failed to germinate or exhibited stunted growth were removed fromthe trial. Stunting was defined as lacking a fully expanded true leaf attime of data collection or having an expanded true leaf judged by eye tobe <½ the average leaf area of the treatment group. Generally, 30 seedswere planted per treatment group and 15-25 plants were used for datacollection.

Root growth was estimated by counting the number of times that a primaryroot crosses the quadrant marks on the bottom of the cup. These wereoften observed along the bottom circumference of the cup, although somewere visible along the side of the container and were counted as ifcrossing a vertical extension of the quadrant line. This number wasdivided by 4 to produce a root growth index. This index was found tocorrelate ˜90% with measured total primary root length (sum of lengthsof all primary roots after rinsing soil from roots and measuringdirectly).

The results of these studies are summarized in Table 20 below. Eachnumber indicates the percentage increase over the untreated controlvalues. An asterisk indicates that the difference was statisticallysignificant with p<0.05 as determined by Student's T-test.

TABLE 20 Summary of Root & Shoot Growth Peptide (Host) SEQ ID NO: Rate(μg/ml) Root Shoot P4-14S-9A (corn) 57 0.2 4.8 3.9%* P4-14S-9A (soy) 575.0 2.5% 6.5%* P4-14S-9A (soy) 57 2.0 4.3% 5.0% P4-14S-16S (soy) 55 2.028.3%* −11.3% P4-14S-16S (soy) 55 0.2 28.3% −2.0% P4-14S-20S (soy) 565.0 2.2% 5.8%* P14 (soy) 113 5.0 24.9%* 4.4% P14a (soy) 114 5.0 10.3%*−3.5% P14a (soy) 114 0.2 −2.7% 8.7%*

Increased root growth (shown in p4-14s-16s, p14, and p14a) increases thewater-gathering ability of the plant and will increase drought andflooding resistance. Conversely, increased shoot growth (as inp4-14s-9a, p4-14s-20s, and p14a soy at 0.2 μg/ml) increaseslight-gathering and energy conversion. It is notable that p14a can causeincreases in either root or shoot growth depending on dosage. P4-14s-16scauses a strong shift in resource allocation to root growth.

Example 4 Growth Tests

Soy seeds (or corn seeds, as indicated) were planted in flats with 2seeds per cell within the flat at a greenhouse facility. The seeds wereallowed to germinate and the smaller plant is culled, leaving one plantper cell. Once the first true leaves are fully expanded and the secondleaves are beginning to expand, the plants were initially measured forheight. This was performed by stretching the highest leaf upward andmeasuring the distance to the soil. Peptides were dissolved in water atthe indicated concentrations (Table 21 below). The plants were thentreated with a foliar spray using widely available spray bottles untilliquid was dripping from the leaves. Six flats of 14 plants each weretreated per condition (peptide or control). Designated untreated controlplants were sprayed with water. The plants were allowed to grow for 14days. The height of the plants was again measured and compared to theoriginal height to quantify growth. Finally, the plants were harvestedby removing the shoots (all above-ground material). For someexperiments, this was weighed upon harvesting to determine the freshmass (including water weight). The shoots were then dried at 70° C. for72 hours, and again weighed to determine dry biomass. Although thismethod uses a similar measure (growth) as in Example 3 above, thetreatment method is different (seed soak vs. foliar spray). As a result,the experimental outcomes may diverge.

TABLE 21 Summary of Growth Measurements Growth SEQ ID Rate increase Drybiomass Fresh mass Peptide NO: (ug/ml) (%) increase (%) increase (%)P1-29 (corn) 130 2.0   7.8  5.7 5.1 P4-14S-9A 57 2.0 11.9% 10.8% ND P14113 2.0 25.8%  1.6% ND P14 113 5.0 31.4%   3% ND P4-14S-12D 59 5.0 −4.2%−3.6% ND; resistant to wilting P14a 114 5.0  8.9% 10.6 83.3% P14a 1142.0  3.4% 11.0% 57.9% P14a 114 0.2 14.7% 12.6% 46.8% P14-32 124 5.012.5%   14% 33.3% P14-32 124 2.0  1.7%  8.2% 27.6% P14-32 124 0.2  9.5% 6.8% 2.9% P14-dc4 123 5.0  −17% −7.6% −15.4% P14-dc4 123 2.0 −4.6 −1.6%0.4% P14-dc4 123 0.2  2.6%  5.6% 7.5% ND: Not determinedSeveral of the studied peptides cause increases in growth and drybiomass (p4-14s-9a, p14, p14a, and p14-32). In addition, severalpeptides caused increased conservation of water as compared with UTCplants which suffered drought stress at trial completion. Conservedwater content was indicated by >10% increase in fresh mass, such asfound in p14a- and p14-32-treated plants. The growth effect was notlimited to soy; corn also exhibited a modest growth benefit upon peptidetreatment with p1-29.

Notably, P14-dc4, which incorporates a truncated C-terminus, seems tolose the significant growth, dry biomass, and fresh mass benefitsobserved for p14. By comparison, removal of the P14 N-terminal sequence(P14a samples) still results in increases for all 3 measures.

Example 5 Induction of Resistance to Tobacco Mosaic Virus

Peptides were tested for the induction of resistance to tobacco mosaicvirus (TMV) in tobacco. Briefly, three tobacco plants at 6-8 weeks oldwere selected per group (samples and controls). The bottom-most leaf ofthe plant was covered and the plant was sprayed with a solution of water(negative control), peptide, or Proact (positive control). The spray wasapplied until the leaves were fully wetted, indicated by liquid drippingfrom the leaves. The plants were then allowed to dry and the leafcovering was removed.

Three days post-treatment, the previously-covered leaf and a leaf on theopposite side of the plant were then lightly dusted with diatomaceousearth and 20 ul of a 1.7 ug/ml solution of purified tobacco mosaic viruswas applied. The TMV solution was then spread across the leaf surface bylightly rubbing solution and the diatomaceous earth across the surfaceof the leaves. Two minutes after inoculation, the diatomaceous earth wasrinsed off the leaves with water. 3 days after TMV inoculation, theleaves were scored based on the number of TMV lesions observed. The leafwas also scored for signs of the hypersensitive response, includingyellowing and wilting of the affected leaves.

Effectiveness described in Table 22 refers to the % decline in TMVlesions on treated vs UTC plants. A reduction of TMV on covered leavesindicates a systemic immune response in the plant while reduction onuncovered leaves indicates a local response. Asterisks indicate that theP-value derived from a T-test was <0.05.

TABLE 22 Summary of TMV Resistance Effectiveness SEQ ID ConcentrationUncovered Effectiveness Peptide NO: (ug/ml) (%) Covered (%) P1-29 130 1044  18  P1-31 131 20 95* 88* P3-5 156 10 66  94* P3-10 159 20 94* 95*P4-14S-9A 57 5.0 84* 86* P4-14S-9D 58 10 89  52  P4-14S-12D 59 5.0 61*60* P4-14S-13Q 54 10 40  61* P4-14S-16A 60 10 1 3 P4-14S-16S 55 10 72*46  P4-14S-16V 135 10.0 78  85* P4-14S-17F 136 10 94  57  P4-14S-20S 5610 59* −45  P4-14S-20V 137 10 −26  −29  P4-14S-21D 50 10 49  48  P4-111132 10 58  65  P4-115 139 20 87* 87* P4-116 140 10 52   0* P4-d18 51 1065  15  P4-d10,14,18 62 10 55  37  P4-i10a 65 10 92  81  P4-i14a 66 1075  77  P4-i18a 67 10 81  50  P4-i10,14,18a 68 10 75  69  P14-22L,26L115 20 65* 67* P14-22L,26E 19 20 70* 91* P14-22A,26L 20 20 48  62 P14-22A,26E 21 20 91* 85* P14-22E,26L 22 20 61* 84* P14-22E,26E 23 2074* 62  P14-22E,26A 25 20 89* 88* P14-22L,26A 26 20 72* 83*P14-22L,25-26A 28 10 87* 75* P14-22L,28-29A 29 10 83* 84* P14-22L,32-33A 30 10 91* 69* P14-22,26A 27 10 92* 93* P14-22L,26,29A 31 10 85*70* P14-22L,28,32A 32 10 71* 37* P14-31 129 20 85* 87* P14-32 124 20 90*82* P14-dc1 121 10 65* 46  P14-dc2 122 10 76* 56  P14-dc4 123 10 34  19 P14-39 160 20 72* 66* P14-41 245 20 86* 61* P14d-7D 208 20 71* 55*P14d-10D 209 20 84* 84* P14d-11D 210 20 81* 87* P14d-14D 211 20 84* 35 P14d-15D 212 20 87* 74* P14d-17D 213 20 68* 65* P14d-18D 214 20 52  32 P14d-7S 215 20 −112   23  P14f-17S 45 20 94* 84* P6a-15D 199 20 74* 74*P6a-11S 204 20 71* 88* P15b-12S 188 20 76* 47  P15b-21S 193 20 100*  97*P18min-11S 94 20 −90  −274   P18min-7D 85 20 26  29  P19min-10S 107 2081* 94* P19min-11S 108 20 71* 45  P19min-13S 109 20 35  −70  P25min-15S81 20 81* 74* P25-9D 224 20 78* 55  P15-60 150 20 55  13  P15-61 151 2055* 7 P15-62 148 20 94* −122   P15-63 149 20 90* 74* P18-8 142 20 55 78* P18-9 143 20 65* 52  P25-9 152 20 68  87* P25-12 153 20 77* 58 P25-14 155 20 52  −87 

Several HR-negative peptides cause significant local and systemicresponses: P4-14S-9A, P4-14S-17A, P4-14S-12D, and P4-14S-16V producedthe best results. In general, most peptides exhibited some anti-TMVactivity. However, peptides with a larger C-terminal truncation ascompared with an intact HR-box (see co-pending U.S. patent applicationSer. No. 14/872,298, entitled “Hypersensitive Response Elicitor Peptidesand Use Thereof”, filed Oct. 1, 2015, which is hereby incorporated byreference in its entirety) (e.g., p4-116, P14-dc4), exhibit reducedeffectiveness against TMV infection. A shorter C-terminal truncationgenerally elicited TMV resistance at a slightly weaker rate (50-75%control). Greater disruption of residue spacing in P4-d10,14,18 alsoexhibited a moderate reduction in effectiveness against TMV infection.Also, mutation of some leucine positions seemed associated with weakerTMV responses: P19min-135, P18min-7D, P18min-11S, P14d-18D, P4-14S-20V,P4-14S-20S, P4-14s-16A. Notably, although it contains a severalhydrophobic sequences, P25-14 exhibits weaker control of TMV infection.A systematic investigation of mutation of the important leucine andisoleucine residues over several HR-eliciting sequences revealed thatmutations at the several locations can cause a reduction in the immuneresponse. Mutation of the first hydrophobic residue (in p14d-7S,p18min-7D, and p25-9D) can cause reduced efficacy on covered leaves,indicating a reduction in systemic immune response. P15b-12S alsoexhibits a similar reduction in systemic responses in the covered leaf.P19min-13S exhibits poor immune activation. One can remove the lasthydrophobic doublet, which still allowed for anti-TMV activity (forexample in P1-31 and P1-111). However, a further shortened hydrophobicsequence is associated with a reduction in activity against TMV, asdemonstrated by P4-116 and P14-dc4.

Having thus described the basic concept of the invention, it will berather apparent to those skilled in the art that the foregoing detaileddisclosure is intended to be presented by way of example only, and isnot limiting. Various alterations, improvements, and modifications willoccur and are intended to those skilled in the art, though not expresslystated herein. These alterations, improvements, and modifications areintended to be suggested hereby, and are within the spirit and scope ofthe invention. Additionally, the recited order of processing elements orsequences, or the use of numbers, letters, or other designationstherefore, is not intended to limit the claimed processes to any orderexcept as may be specified in the claims. Accordingly, the invention islimited only by the following claims and equivalents thereto.

What is claimed:
 1. An isolated peptide comprising the amino acidsequence of L-X-X-(L/I)-(L/I)-X-X-(L/I/V)-(L/I/V) (SEQ ID NO: 116),wherein the peptide is free of cysteine and methionine; each X atpositions 2, 3, 6, 7 is independently selected from the group consistingof G, A, S, T, D, isoD, E, γ-glutamate Q, N, K and R; the peptidecomprises up to 24 amino acids at the N-terminal end of SEQ ID NO: 116,up to 10 amino acids at the C-terminal end of SEQ ID NO: 116, or both,except that the amino acid sequence of the peptide does not comprise(L/I/V/F)-X-X-(L/I/V/F)-(L/I)-X-X-(L/I/V)-(L/I)-X-X-(L/I/V/F)-(L/I/V/F)(SEQ ID NO: 125) where each X at positions 2, 3, 6, 7, 10, 11 is anyamino acid; and the peptide induces an active plant response, but doesnot induce a hypersensitive response, when applied to mechanicallydisrupted plant tissue.
 2. The isolated peptide according to claim 1,wherein the peptide comprises up to 20 amino acids at the N-terminal endof SEQ ID NO: 116, up to 7 amino acids at the C-terminal end of SEQ IDNO: 116, or both.
 3. The isolated peptide according to claim 1, whereinthe peptide comprises the amino acid sequence of one of SEQ ID NOS: 124,129-132, 139, 143, and
 160. 4. The isolated peptide according to claim1, wherein the peptide consists essentially of the amino acid sequenceof one of SEQ ID NOS: 124, 129-132, 139, 143, and
 160. 5. The isolatedpeptide according to claim 1, wherein the isolated peptide ischaracterized by one or more of: (i) stable when dissolved in water oraqueous solution, (ii) resistant to chemical degradation when dissolvedin an aqueous buffer solution containing a biocide, (iii) solubility ofgreater than about 0.1% in water or aqueous solution.
 6. The isolatedpeptide according to claim 1, wherein the active plant response inducedby the peptide is selected from the group consisting of peroxideinduction, enhanced growth, pathogen resistance, biotic or abioticstress resistance, and modified biochemical signaling.
 7. The isolatedpeptide according to claim 1, wherein the peptide is at least 90% pure.8. The isolated peptide according to claim 1, wherein the peptide is afusion polypeptide comprising a second amino acid sequence coupled viapeptide bond to the amino acid sequence.
 9. The isolated peptideaccording to claim 8, wherein the second amino acid sequence includes apurification tag.
 10. The isolated peptide according to claim 9, whereinthe second amino acid sequence further includes a cleavable linkersequence between the purification tag and the amino acid sequence. 11.The isolated peptide according to claim 8, wherein the peptide is afusion polypeptide comprising a first amino acid sequence for saidpeptide linked to a second amino acid sequence for said peptide.
 12. Afusion polypeptide comprising a plurality of amino acid sequences linkedtogether in series, one of the plurality of amino acid sequencescomprising a peptide according to claim
 1. 13. A composition comprisingone or more peptides according to claim 1 and a carrier.
 14. Thecomposition according to claim 13, wherein the composition is aclarified cell extract.
 15. The composition according to claim 13further comprising an additive selected from the group consisting offertilizer, herbicide, insecticide, fungicide, nematicide, abactericidal agent, a biological inoculant, a plant regulator, andmixtures thereof.
 16. The composition according to claim 15, wherein theadditive comprises either: (i) clothianidin, a combination ofclothianidin and Bacillus firmus, imidicloprid, or a combination ofimidicloprid and Bacillus firmus; or (ii) thiamethoxam; a combination ofthiamethoxam, mefenoxam, and fludioxynil; a combination of thiamethoxam,mefenoxam, fludioxynil and azoxystrobin; a combination of thiamethoxamand abamectin; a combination of thiamethoxam, abamectin, and a Pasteurianematicide; or a combination of thiamethoxam, mefenoxam, fludioxynil,azoxystrobin, thiabendazole, and abamectin; or (iii) a biologicalinoculant comprising a Bradyrhizobium spp., a Bacillus spp., or acombination of a Bradyrhizobium spp. and a Bacillus spp.
 17. Thecomposition according to claim 13, wherein the carrier is an aqueouscarrier.
 18. The composition according to claim 17, wherein the aqueouscarrier further comprises one or more of a biocidal agent, a proteaseinhibitor, a non-ionic surfactant, or a combination thereof.
 19. Thecomposition according to claim 13, wherein the carrier is a solidcarrier in particulate form.
 20. The composition according to claim 19,wherein the solid carrier is a dry powder.
 21. The isolated peptideaccording to claim 1, wherein the amino acid sequence of the peptidecontains no lysine or arginine residues except optionally at theC-terminal end.
 22. The isolated peptide according to claim 1, whereineach X at positions 2, 3, 6, 7 is independently selected from the groupconsisting of G, A, S, T, D, isoD, E, y-glutamate, Q, and N, and whereinthe peptide further comprises an arginine residue at the C-terminal endof the peptide.
 23. The isolated peptide according to claim 1, whereinthe peptide comprises the amino acid sequence of one of SEQ ID NOS: 124,129, 130, and 132, except that (i) any lysine or arginine residues arechanged to glutamate and (ii) an arginine residue is introduced at theC-terminal end of the peptide.
 24. The isolated peptide according toclaim 1, wherein the peptide comprises the amino acid sequence of one ofSEQ ID NOS: 131, 139, 143, and 160, and the peptide further comprises anarginine residue at the C-terminal end of the peptide.
 25. An isolatedpeptide that is up to 50 amino acids in length and comprises the aminoacid sequence of: (a) one of SEQ ID NOS: 115, 124, 128-132, 139, 143,146, 147, 151, 152, and 160; or (b) one of SEQ ID NOS: 115, 124, 129,130, 132, 146, 147, 151, and 152 except that (i) any lysine or arginineresidues are changed to glutamate and (ii) an arginine residue isintroduced at the C-terminal end of the peptide; wherein the peptideinduces an active plant response, but does not induce a hypersensitiveresponse, when applied to mechanically disrupted plant tissue.
 26. Theisolated peptide according to claim 25, wherein the peptide consistsessentially of the amino acid sequence of one of SEQ ID NOS: 115, 124,128-132, 139, 143, 146, 147, 151, 152, and
 160. 27. The isolated peptideaccording to claim 25, wherein the peptide comprises the amino acidsequence of one of SEQ ID NOS: 128, 131, 139, 143, and 160, and thepeptide further comprises an arginine residue at the C-terminal end ofthe peptide.
 28. A fusion polypeptide comprising a plurality of aminoacid sequences linked together in series, one of the plurality of aminoacid sequences comprising the peptide according to claim
 25. 29. Acomposition comprising one or more peptides according to claim 25 and acarrier.
 30. A method of imparting disease resistance to plantscomprising: applying an effective amount of an isolated peptideaccording to claim 25 to a plant or plant seed or the locus where theplant is growing or is expected to grow, wherein said applying iseffective to impart disease resistance.
 31. A method of enhancing plantgrowth comprising: applying an effective amount of an isolated peptideaccording to claim 25 to a plant or plant seed or the locus where theplant is growing or is expected to grow, wherein said applying iseffective to enhance plant growth.
 32. A method of increasing a plant'stolerance to biotic stress comprising: applying an effective amount ofan isolated peptide according to claim 25 to a plant or plant seed orthe locus where the plant is growing or is expected to grow, whereinsaid applying is effective to increase the plant's tolerance to bioticstress factors selected from the group consisting of insects, arachnids,nematodes, weeds, and combinations thereof.
 33. A method of increasing aplant's tolerance to abiotic stress comprising: applying an effectiveamount of an isolated peptide according to claim 25 to a plant or plantseed or the locus where the plant is growing or is expected to grow,wherein said applying is effective to increase the plant's tolerance toabiotic stress factors selected from the group consisting of saltstress, water stress, ozone stress, heavy metal stress, cold stress,heat stress, nutritional stress, and combinations thereof.
 34. A methodof imparting disease resistance to plants comprising: applying aneffective amount of an isolated peptide according claim 1 to a plant orplant seed or the locus where the plant is growing or is expected togrow, wherein said applying is effective to impart disease resistance.35. A method of enhancing plant growth comprising: applying an effectiveamount of an isolated peptide according to claim 1 to a plant or plantseed or the locus where the plant is growing or is expected to grow,wherein said applying is effective to enhance plant growth.
 36. A methodof increasing a plant's tolerance and resistance to biotic stresscomprising: applying an effective amount of an isolated peptideaccording to claim 1 to a plant or plant seed or the locus where theplant is growing or is expected to grow, wherein said applying iseffective to increase the plant's tolerance and resistance to bioticstress factors selected from the group consisting of insects, arachnids,nematodes, weeds, and combinations thereof.
 37. A method of increasing aplant's tolerance to abiotic stress comprising: applying an effectiveamount of an isolated peptide according to claim 1 to a plant or plantseed or the locus where the plant is growing or is expected to grow,wherein said applying is effective to increase the plant's tolerance toabiotic stress factors selected from the group consisting of saltstress, water stress, ozone stress, heavy metal stress, cold stress,heat stress, nutritional stress, and combinations thereof.